Host cell capable of producing enzymes useful for degradation of lignocellulosic material

ABSTRACT

The invention relates to a host cell comprising at least four different heterologous polynucleotides chosen from the group of polynucleotides encoding cellulases, hemicellulases and pectinases, wherein the host cell is capable of producing the at least four different enzymes chosen from the group of cellulases, hemicellulases and pectinases, wherein the host cell is a filamentous fungus and is capable of secretion of the at least four different enzymes. This host cell can suitably be used for the production of an enzyme composition that can be used in a process for the saccharification of cellulosic material.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 13/578,152, filed Oct.24, 2012, which is a 371 of PCT/EP2011/052059, filed Feb. 11, 2011,which claims priority to EP 10153353.7, filed Feb. 11, 2010, thecontents of which are incorporated herein by reference in theirentireties.

STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

This invention was made with US Government support under Grant No.DE-FC36-08G018079, awarded by the Department of Energy. The USGovernment may have certain rights in this invention.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

A Sequence Listing is submitted herewith as an ASCII compliant text filenamed “2919208-198001_(—) Sequence_listing.txt”), created on Jun. 23,2015, and having a size of 48,485 bytes as permitted under 37 C.F.R.§1.821(c). The material in the aforementioned file is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates to a host cell capable of producing at least fourdifferent enzymes chosen from the group of cellulases, hemicellulasesand pectinases, a process for the preparation of said host cell, anenzyme composition produced by said host cell, a process for theproduction of an enzyme composition using said host cell, a process forthe saccharification of lignocellulosic material using the enzymecomposition and a process for the preparation of a fermentation productusing the enzyme composition.

Description of Related Art

Carbohydrates constitute the most abundant organic compounds on earth.However, much of this carbohydrate is sequestered in complex polymersincluding starch (the principle storage carbohydrate in seeds andgrain), and a collection of carbohydrates and lignins known aslignocellulose. The main carbohydrate components of lignocellulose arecellulose, hemicellulose, and pectins. These complex polymers are oftenreferred to collectively as lignocellulose.

Bioconversion of renewable lignocellulosic biomass to a fermentablesugar that is subsequently fermented to produce alcohol (e.g., ethanol)as an alternative to liquid fuels has attracted an intensive attentionof researchers since 1970s, when the oil crisis broke out because ofdecreasing the output of petroleum by OPEC. Ethanol has been widely usedas a 10% blend to gasoline in the USA or as a neat fuel for vehicles inBrazil in the last two decades. More recently, the use of E85, an 85%ethanol blend has been implemented especially for clean cityapplications. The importance of fuel bioethanol will increase inparallel with increases in prices for oil and the gradual depletion ofits sources. Additionally, fermentable sugars are being used to produceplastics, polymers and other biobased products and this industry isexpected to grow substantially therefore increasing the demand forabundant low cost fermentable sugars which can be used as a feed stockin lieu of petroleum based feedstocks.

The sequestration of large amounts of carbohydrates in plant biomassprovides a plentiful source of potential energy in the form of sugars,both five carbon and six carbon sugars that could be utilized fornumerous industrial and agricultural processes. However, the enormousenergy potential of these carbohydrates is currently under-utilizedbecause the sugars are locked in complex polymers, and hence are notreadily accessible for fermentation. Methods that generate sugars fromplant biomass would provide plentiful, economically-competitivefeedstocks for fermentation into chemicals, plastics, and fuels.

Regardless of the type of cellulosic feedstock, the cost and hydrolyticefficiency of enzymes are major factors that restrict thecommercialization of the biomass bioconversion processes. The productioncosts of microbially produced enzymes are tightly connected with aproductivity of the enzyme-producing strain.

With productivity of a strain is meant the amount of total protein orenzyme activity produced in the fermentation broth per time, for examplekg protein/(m³ broth.year) or enzyme units/(m³ broth.year).

An enzyme unit is the ability of a defined amount of protein to converta defined substrate under certain defined conditions.

SUMMARY

The presence of a separate ‘cellulase inducer’ is usually needed toproduce the enzymes for biomass conversion in a microorganism. Acellulase inducer is disadvantageous for the following reasons. First,the inducer, such as a plant material, may have a variable composition,which is disadvantageous for the controllability of the expression ofthe heterologous polynucleotides chosen from the group ofpolynucleotides encoding cellulases, hemicellulases and pectinases.Secondly, energy is required to sterilise plant material before theinducer can be used for inducing the production of the at least fourdifferent enzymes chosen from the group of cellulases, hemicellulase andpectinases. Thirdly, plant material will heavily pollute fermentationequipment. Fourthly, the inducer may result in a higher viscosity of themedium wherein the host cell expresses the at least four differentpolynucleotides chosen from the group of polynucleotides encodingcellulases, hemicellulases and pectinases. Fifthly, the presence of a‘cellulase inducer’, in particular when it has been pre-treated, mayresult in the production of inhibitors that may be detrimental to thehost cell.

“Cellulase inducer” is herein defined as a compound that induces theproduction of cellulase in the host cell. Examples of cellulase inducersinclude pure cellulose, cellobiose, sophorose and gentiobiose or anylignocellulosic material.

It would therefore be highly desirable to provide a simple andeconomically attractive enzymatic process for the production of sugarsfrom plant biomass.

This object has been achieved by the provision of a host cell comprisingat least four different heterologous polynucleotides chosen from thegroup of polynucleotides encoding cellulases, hemicellulases andpectinases, wherein the host cell is capable of producing the at leastfour different enzymes chosen from the group of cellulases,hemicellulases and pectinases, wherein the host cell is a filamentousfungus and is capable of secretion of the at least four differentenzymes

Since all enzymes necessary for degradation of lignocellulosic materialare produced in and secreted by one and the same host cell, aready-to-use enzyme composition can be produced in only onefermentation. The enzyme composition produced by the host cell of theinvention can suitably be used in a process for the saccharification oflignocellulosic material.

Furthermore, the host cell of the invention may not need a cellulaseinducer to produce the at least four different enzymes chosen from thegroup of cellulases, hemicellulases and pectinases.

A possible advantage of using the host cell of the invention in aprocess for the saccharification of lignocellulosic material is that itis not necessary to blend/mix the enzymes needed for the degradation ofthe lignocellulosic material in the desired ratios. Furthermore, thereis no need to store separate enzymes before using them in a process forthe degradation of lignocellulosic material.

Furthermore, by selecting the host cell and the genetic modifications ofthe host cell according to the preferred embodiments of the invention, ahigh productivity of the enzymes can be achieved.

Furthermore, the fermentation broth (comprising the at least fourdifferent enzymes chosen from the group of cellulases, hemicellulasesand pectinases) may be used without any purification in a process forthe degradation of lignocellulosic material. Therefore, the inventionprovides a very simple and cost-effective solution.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Map of pGBTOPEBA205 for expression of T. emersoniicellobiohydrolase I (CBH I) in T. emersonii. Depicted are EBA205expressed from the glucoamylase promoter (PglaA). In addition, theglucoamylase flank (3′glaA) of the expression cassette is depicted.pGBTOPEBA205 is representative for pGBTOPEBA8, which comprises theDNA-sequence encoding the T. emersonii EG, and pGBTOPEBA4, whichcomprises DNA-sequence encoding the T. emersonii BG.

FIG. 2: Map of pGBFINEBA176 for expression of T. emersoniicellobiohydrolase II (CBHII) in T. emersonii. pGBFINEBA176 is apGBFIN11-based plasmid. Depicted is EBA176 expressed from theglucoamylase promoter (PglaA). In addition, the selectable marker(amdS), expressed from the Aspergillus nidulansglyceraldehyde-3-phosphate dehydrogenease promoter (Pgpd) and theglucoamylase flanks (3′glaA and 3″glaA) of the expression cassette aredepicted.

FIGS. 3A-3B: Detection of multiple recombinant T. emersonii cellulasesin A. niger.

(3A). SDS-PAGE detection of T. emersonii cellulases expressed in A.niger. A. niger was transformed with a mix of pGBTOPEBA4, pGBTOPEBA8,pGBFINEBA176, and pGBTOPEBA205. Transformants were grown in shake flaskscontaining maltose containing medium and proteins in supernatantsharvested from 96 hours cultures were analysed by SDS-PAGE analysis.

(3B). Graph showing WSU activity in transformants. Transformants weregrown for 96 hours in maltose containing medium and WSU activity wasdetermined in supernatants of the cultures.

FIGS. 4A-4B: Detection of multiple recombinant T. emersonii cellulasesin T. emersonii.

(4A). SDS-PAGE detection of T. emersonii cellulases expressed in T.emersonii. T. emersonii was transformed with a mix of pGBTOPEBA4,pGBTOPEBA8, pGBFINEBA176, and pGBTOPEBA205. Approximately 400transformants were grown in 96-well plates and screened for expressionof at least one cellulase by E-PAGE gel analysis. Interestingtransformants were grown in shake flasks containing glucose-based mediumand proteins in supernatants harvested from 72 hours cultures wereTCA-precipitated and analysed by SDS-PAGE analysis. FBG142 is the emptystrain.

(4B). Graph showing WSU activity in transformants. Transformants weregrown for 72 hours in glucose-based medium and WSU activity wasdetermined in 16-times diluted supernatants of the cultures. FBG142 isthe empty strain.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 sets out the amino acid sequence of a cellobiohydrolase Ifrom Talaromyces emersonii

SEQ ID NO: 2: sets out the polynucleotide from Talaromyces emersoniiencoding the amino acid sequence of SEQ ID NO. 1.

SEQ ID NO: 3 sets out the amino acid sequence of a β-glucanase CEA fromTalaromyces emersonii.

SEQ ID NO: 4 sets out the polynucleotide from Talaromyces emersoniiencoding the amino acid sequence of SEQ ID NO. 3.

SEQ ID NO: 5 sets out the amino acid sequence of a β-glucosidase fromTalaromyces emersonii.

SEQ ID NO: 6 sets out the polynucleotide from Talaromyces emersoniiencoding the amino acid sequence of SEQ ID NO. 5.

SEQ ID NO: 7 sets out the amino acid sequence of a cellobiohydrolase IIfrom Talaromyces emersonii.

SEQ ID NO: 8 sets out the polynucleotide from Talaromyces emersoniiencoding the amino acid sequence of SEQ ID NO. 7.

SEQ ID NO: 9 sets out the aminoacid sequence of a Size 209 aa unknownprotein from T. emersonii.

SEQ ID NO: 10 sets out the coding sequence of an unknown protein from T.emersonii having aminoacid sequence according to SEQ ID NO: 9.

SEQ ID NO: 11 sets out the aminoacid sequence of T. emersonii swollenin.

SEQ ID NO: 12 sets out the coding sequence of T. emersonii swollenin.

SEQ ID NO: 13 sets out the aminoacid sequence of T. emersonii acetylxylan esterase.

SEQ ID NO: 14 sets out the coding sequence of T. emersonii acetyl xylanesterase.

SEQ ID NO: 15 sets out the aminoacid sequence of T. emersonii xylanase.

SEQ ID NO: 16 sets out the coding sequence of T. emersonii xylanase.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT

As used herein the expression “heterologous polynucleotides” meanspolynucleotides that are not present in the untransformed host cell,whereas “homologous polynucleotides” means polynucleotides that arepresent in the untransformed host cell. Synthetic polynucleotides areherein considered heterologous polynucleotides, independent of theirpotential presence in the untransformed host cell.

As used herein “transformant” means a cell that has been the object oftransformation. “Transformant”, “host cell” and “recombinant cell” areherein used as synonyms.

“Transformation” herein means the genetic alteration of a cell by meansof recombinant technology. It may result in the uptake, incorporation,and expression of genetic material (DNA, RNA or protein) or mutation ordeletion of genetic material in the cell, through human intervention.

The Host Cell

The host cell according to the invention may be prepared from any cell.For specific uses of a compound produced in a host cell according to theinvention, the selection of the cell to be transformed may be madeaccording to such use. Where e.g. the compound produced in a host cellaccording to the invention is to be used in food applications, a hostcell may be selected from a food-grade organism such as Saccharomycescerevisiae. Specific uses include, but are not limited to, food,(animal) feed, pharmaceutical, agricultural such as crop-protection,and/or personal care applications.

According to one embodiment, the host cell according to the invention isa eukaryotic host cell. Preferably, the eukaryotic cell is a mammalian,insect, plant, fungal, or algal cell. Preferred mammalian cells includee.g. Chinese hamster ovary (CHO) cells, COS cells, 293 cells, PerC6™cells, and hybridomas. Preferred insect cells include e.g. Sf9 and Sf21cells and derivatives thereof. More preferably, the eukaryotic cell is afungal cell, i.e. a yeast cell, such as a Candida, Hansenula,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiastrain, more preferably from Kluyveromyces lactis, S. cerevisiae,Hansenula polymorpha, Yarrowia lipolytica and Pichia pastoris, or afilamentous fungal cell. Most preferably, the eukaryotic cell is afilamentous fungal cell.

“Filamentous fungi” include all filamentous forms of the subdivisionEumycota and Oomycota (as defined by Hawksworth et al., In, Ainsworthand Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK). The filamentous fungiare characterized by a mycelial wall composed of chitin, cellulose,glucan, chitosan, mannan, and other complex polysaccharides. Vegetativegrowth is by hyphal elongation and carbon catabolism is obligatelyaerobic. Filamentous fungal strains include, but are not limited to,strains of Acremonium, Agaricus, Aspergillus, Aureobasidium,Chrysosporium, Coprinus, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Piromyces, Panerochaete, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, andTrichoderma.

Several strains of filamentous fungi are readily accessible to thepublic in a number of culture collections, such as the American TypeCulture Collection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL). Examples of such strains includeAspergillus niger CBS 513.88, Aspergillus oryzae ATCC 20423, IFO 4177,ATCC 1011, ATCC 9576, ATCC14488-14491, ATCC 11601, ATCC12892, P.chrysogenum CBS 455.95, Penicillium citrinum ATCC 38065, Penicilliumchrysogenum P2, Talaromyces emersonii CBS 393.64, Acremonium chrysogenumATCC 36225 or ATCC 48272, Trichoderma reesei ATCC 26921 or ATCC 56765 orATCC 26921, Aspergillus sojae ATCC11906, Chrysosporium lucknowenseATCC44006 and derivatives thereof.

Preferred filamentous fungal cells belong to Aspergillus, Chrysosporium,Penicillium, Talaromyces or Trichoderma genus, and most preferablyAspergillus niger, Aspergillus awamori, Aspergillus foetidus,Aspergillus sojae, Aspergillus fumigatus, Talaromyces emersonii,Aspergillus oryzae, Chrysosporium lucknowense, Trichoderma reesei orPenicillium chrysogenum.

Most preferably, the cell to be transformed to form the host cell is anAspergillus.

An advantage of expression of the enzymes (the at least four differentcellulases, hemicellulases and/or pectinases) in Aspergillus may be ahigh enzyme yield on sugar.

When the cell to be transformed to form the host cell according to theinvention is an Aspergillus strain, the Aspergillus strain is preferablyCBS 513.88 or a derivative thereof, more preferably the host cell isAspergillus niger CBS 513.88.

According to another embodiment, the cell to be transformed to form thehost cell according to the invention is a prokaryotic cell. Preferably,the prokaryotic host cell is bacterial cell. The term “bacterial cell”includes both Gram-negative and Gram-positive microorganisms. Suitablebacteria may be selected from the group of Escherichia, Anabaena,Caulobactert, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus,Bacillus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium),Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus,Methylobacterium, Staphylococcus and Streptomyces. Preferably, thebacterial cell is selected from the group of B. subtilis, B.amyloliquefaciens, B. licheniformis, B. puntis, B. megaterium, B.halodurans, B. pumilus, G. oxydans, Caulobactert crescentus CB 15,Methylobacterium extorquens, Rhodobacter sphaeroides, Pseudomonaszeaxanthinifaciens, Paracoccus denitrificans, E. coli, C. glutamicum,Staphylococcus carnosus, Streptomyces lividans, Sinorhizobium meliotiand Rhizobium radiobacter.

The Enzymes

In the invention, the at least four different enzymes are chosen fromthe group of cellulases, hemicellulases and pectinases, preferably fromthe group of cellulases and hemicellulases and more preferably from thegroup of cellulases.

Cellulases are enzymes that hydrolyze cellulose (β-1,4-glucan or βD-glucosidic linkages) resulting in the formation of glucose,cellobiose, cellooligosaccharides, and the like.

Cellulases have been traditionally divided into three major classes:endoglucanases (EC 3.2.1.4) (“EG”), exoglucanases or cellobiohydrolases(EC 3.2.1.91) (“CBH”) and β-glucosidases ([β]-D-glucosideglucohydrolase; EC 3.2.1.21) (“BG”). See e.g. Knowles et al., TIBTECH 5,255-261, 1987; Shulein, Methods Enzymol., 160, 25, pp. 234-243, 1988.Endoglucanases act mainly on the amorphous parts of the cellulose fibre,whereas cellobiohydrolases are also able to degrade crystallinecellulose (Nevalainen and Penttila, Mycota, 303-319, 1995). Thus, thepresence of a cellobiohydrolase in a cellulase system is required forefficient solubilization of crystalline cellulose (Suurnakki, et al.Cellulose 7:189-209, 2000). β-glucosidase acts to liberate D-glucoseunits from cellobiose, cello-oligosaccharides, and other glucosides(Freer, J. Biol. Chem. vol. 268, no. 13, pp. 9337-9342, 1993).

Cellulases are known to be produced by a large number of bacteria, yeastand fungi. Certain fungi produce a complete cellulase system capable ofdegrading crystalline forms of cellulose, such that the cellulases arereadily produced in large quantities via fermentation. Filamentous fungiplay a special role since many yeast, such as Saccharomyces cerevisiae,lack the ability to hydrolyze cellulose. See, e.g., Aubert et al., 1988;Wood et al., Methods in Enzymology, vol. 160, no. 9, pp. 87-116, 1988,and Coughlan, et al., “Comparative Biochemistry of Fungal and BacterialCellulolytic Enzyme Systems” Biochemistry and Genetics of CelluloseDegradation, pp. 11-30 1988.

Fungal cellulase classifications of CBH, EG and BG can be furtherexpanded to include multiple components within each classification. Forexample, multiple CBHs, EGs and BGs have been isolated from a variety offungal sources including Trichoderma reesei which contains knownpolynucleotides coding for 2 CBHs, i.e., CBH I and CBH II, at least 8EGs, i.e., EG I, EG II, EG III, EGIV, EGV, EGVI, EGVII and EGVIII, andat least 5 BGs, i.e., BG1, BG2, BG3, BG4 and BG5.

Accordingly, an enzyme composition of the invention may comprise anycellulase, for example, a cellobiohydrolase, an endo-β-1,4-glucanase, aβ-glucosidase or a β-(1,3)(1,4)-glucanase.

Herein, a cellobiohydrolase (EC 3.2.1.91) is any polypeptide which iscapable of catalysing the hydrolysis of 1,4-β-D-glucosidic linkages incellulose or cellotetraose, releasing cellobiose from the non-reducingends of the chains. This enzyme may also be referred to as cellulase1,4-β-cellobiosidase, 1,4-β-cellobiohydrolase, 1,4-β-D-glucancellobiohydrolase, avicelase, exo-1,4-β-D-glucanase,exocellobiohydrolase or exoglucanase.

Herein, an endo-β-1,4-glucanase (EC 3.2.1.4) is any polypeptide which iscapable of catalysing the endohydrolysis of 1,4-β-D-glucosidic linkagesin cellulose, lichenin or cereal β-D-glucans. Such a polypeptide mayalso be capable of hydrolyzing 1,4-linkages in β-D-glucans alsocontaining 1,3-linkages. This enzyme may also be referred to ascellulase, avicelase, β-1,4-endoglucan hydrolase, β-1,4-glucanase,carboxymethyl cellulase, celludextrinase, endo-1,4-β-D-glucanase,endo-1,4-β-D-glucanohydrolase, endo-1,4-β-glucanase or endoglucanase.

Herein a β-(1,3)(1,4)-glucanase (EC 3.2.1.73) is any polypeptide whichis capable of catalyzing the hydrolysis of 1,4-β-D-glucosidic linkagesin β-D-glucans containing 1,3- and 1,4-bonds. Such a polypeptide may acton lichenin and cereal β-D-glucans, but not on β-D-glucans containingonly 1,3- or 1,4-bonds. This enzyme may also be referred to aslicheninase, 1,3-1,4-β-D-glucan 4-glucanohydrolase, β-glucanase,endo-β-1,3-1,4 glucanase, lichenase or mixed linkage β-glucanase. Analternative for this type of enzyme is EC 3.2.1.6, which is described asendo-1,3(4)-β-glucanase. This type of enzyme hydrolyses 1,3- or1,4-linkages in β-D-glucans when the glucose residue whose reducinggroup is involved in the linkage to be hydrolysed is itself substitutedat C-3. Alternative names include endo-1,3-β-glucanase, laminarinase,1,3-(1,3;1,4)-β-D-glucan 3 (4) glucanohydrolase; substrates includelaminarin, lichenin and cereal β-D-glucans.

Herein, a β-glucosidase (EC 3.2.1.21) is any polypeptide which iscapable of catalysing the hydrolysis of terminal, non-reducingβ-D-glucose residues with release of β-D-glucose. Such a polypeptide mayhave a wide specificity for β-D-glucosides and may also hydrolyze one ormore of the following: a β-D-galactoside, an α-L-arabinoside, aβ-D-xyloside or a β-D-fucoside. This enzyme may also be referred to asamygdalase, β-D-glucoside glucohydrolase, cellobiase or gentobiase.

The host cell may produce and secrete endoglucanase activity and/orcellobiohydrolase activity and/or β-glucosidase activity. The host cellmay produce and secrete more than one enzyme activity in one or more ofthose classes. For example, the host cell may produce and secrete twoendoglucanase activities, for example, endo-1,3(1,4)-β glucanaseactivity and endo-β-1,4-glucanase activity.

Herein, a hemicellulase is any polypeptide which is capable of degradingor modifying hemicellulose. That is to say, a hemicellulase may becapable of degrading or modifying one or more of xylan, araban,glucuronoxylan, arabinogalactan, arabinoxylan, glucomannan,galactomannan and xyloglucan. A polypeptide which is capable ofdegrading a hemicellulose is one which is capable of catalysing theprocess of breaking down the hemicellulose into smaller polysaccharides,either partially, for example into oligosaccharides, or completely intosugar monomers, for example hexose or pentose sugar monomers. Ahemicellulase may give rise to a mixed population of oligosaccharidesand sugar monomers when contacted with the hemicellulase. Suchdegradation will typically take place by way of a hydrolysis reaction.

A composition of the invention may comprise any hemicellulase, forexample, an endoxylanase, a β-xylosidase, an α-L-arabinofuranosidase, anacetyl-xylan esterase, an α-D-glucuronidase, an cellobiohydrolase, aferuloyl esterase, a coumaroyl esterase, an α-galactosidase, aβ-galactosidase, a β-mannanase or a β-mannosidase.

Herein, an endoxylanase (EC 3.2.1.8) is any polypeptide which is capableof catalyzing the endohydrolysis of 1,4-β-D-xylosidic linkages inxylans. This enzyme may also be referred to as endo-1,4-β-xylanase or1,4-β-D-xylan xylanohydrolase. An alternative endoxylanase is EC3.2.1.136, a glucuronoarabinoxylan endoxylanase, an enzyme that is ableto hydrolyse 1,4 xylosidic linkages in glucuronoarabinoxylans.

Herein, a β-xylosidase (EC 3.2.1.37) is any polypeptide which is capableof catalyzing the hydrolysis of 1,4-β-D-xylans, to remove successiveD-xylose residues from the non-reducing termini. Such enzymes may alsohydrolyze xylobiose. This enzyme may also be referred to as xylan1,4-β-xylosidase, 1,4-β-D-xylan xylohydrolase, exo-1,4-δ-xylosidase orxylobiase.

Herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Herein, an α-D-glucuronidase (EC 3.2.1.139) is any polypeptide which iscapable of catalyzing a reaction of the following form:alpha-D-glucuronoside+H₂O→alcohol+D-glucuronate. This enzyme may also bereferred to as alpha-glucuronidase or alpha-glucosiduronase. Theseenzymes may also hydrolyse 4-O-methylated glucoronic acid, which canalso be present as a substituent in xylans. Alternative is EC 3.2.1.131:xylan alpha-1,2-glucuronosidase, which catalyses the hydrolysis ofalpha-1,2-(4-O-methyl)glucuronosyl links.

Herein, an acetyl-xylan esterase (EC 3.1.1.72) is any polypeptide whichis capable of hydrolysis of specifically the ester linkages of theacetyl groups in positions 2 and/or 3 of the xylose moieties of naturalxylan.

Herein, a feruloyl esterase (EC 3.1.1.73) is any polypeptide which iscapable of catalyzing a reaction of the form:feruloyl-saccharide+H₂O→ferulate+saccharide. The saccharide may be, forexample, an oligosaccharide or a polysaccharide. It may typicallycatalyze the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl (feruloyl)group from an esterified sugar, which is usually arabinose in ‘natural’substrates. p-nitrophenol acetate and methyl ferulate are typicallypoorer substrates. This enzyme may also be referred to as cinnamoylester hydrolase, ferulic acid esterase or hydroxycinnamoyl esterase. Itmay also be referred to as a hemicellulase accessory enzyme, since itmay help xylanases and pectinases to break down plant cell wallhemicellulose and pectin.

Herein, a coumaroyl esterase (EC 3.1.1.73) is any polypeptide which iscapable of catalyzing a reaction of the form: coumaroyl-saccharide+H₂O 4coumarate+saccharide. The saccharide may be, for example, anoligosaccharide or a polysaccharide. This enzyme may also be referred toas trans-4-coumaroyl esterase, trans-p-coumaroyl esterase, p-coumaroylesterase or p-coumaric acid esterase. This enzyme also falls within EC3.1.1.73 so may also be referred to as a feruloyl esterase.

Herein, an α-galactosidase (EC 3.2.1.22) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal, non-reducingα-D-galactose residues in α-D-galactosides, including galactoseoligosaccharides, galactomannans, galactans and arabinogalactans. Such apolypeptide may also be capable of hydrolyzing α-D-fucosides. Thisenzyme may also be referred to as melibiase.

Herein, a δ-galactosidase (EC 3.2.1.23) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal non-reducingβ-D-galactose residues in β-D-galactosides. Such a polypeptide may alsobe capable of hydrolyzing α-L-arabinosides. This enzyme may also bereferred to as exo-(1→4)-β-D-galactanase or lactase. β-galactosidase isan enzyme that may accept as its substrate both hemicellulose andpectine, therefore it is classified as a hemicellulase and as apectinase.

Herein, a δ-mannanase (EC 3.2.1.78) is any polypeptide which is capableof catalyzing the random hydrolysis of 1,4-β-D-mannosidic linkages inmannans, galactomannans and glucomannans. This enzyme may also bereferred to as mannan endo-1,4-β-mannosidase or endo-1,4-mannanase.

Herein, a β-mannosidase (EC 3.2.1.25) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal, non-reducingβ-D-mannose residues in β-D-mannosides. This enzyme may also be referredto as mannanase or mannase.

Herein, a pectinase is any polypeptide which is capable of degrading ormodifying pectin. A polypeptide which is capable of degrading pectin isone which is capable of catalysing the process of breaking down pectininto smaller units, either partially, for example into oligosaccharides,or completely into sugar monomers. A pectinase according to theinvention may give rise to a mixed population of oligosaccharides andsugar monomers when contacted with the pectinase. Such degradation willtypically take place by way of a hydrolysis reaction.

A host cell according to the invention may comprise a heterologouspolynucleotide encoding any pectinase, for example an endopolygalacturonase, a pectin methyl esterase, an endo-galactanase, aβ-galactosidase, a pectin acetyl esterase, an endo-pectin lyase, pectatelyase, alpha rhamnosidase, an exo-galacturonase, anexo-polygalacturonate lyase, a rhamnogalacturonan hydrolase, arhamnogalacturonan lyase, a rhamnogalacturonan acetyl esterase, arhamnogalacturonan galacturonohydrolase or a xylogalacturonase.

Herein, an endo-polygalacturonase (EC 3.2.1.15) is any polypeptide whichis capable of catalyzing the random hydrolysis of1,4-α-D-galactosiduronic linkages in pectate and other galacturonans.This enzyme may also be referred to as polygalacturonase pectindepolymerase, endopolygalacturonase, pectolase, pectin hydrolase, pectinpolygalacturonase, poly-α-1,4-galacturonide glycanohydrolase,endogalacturonase; endo-D-galacturonase or poly(1,4-α-D-galacturonide)glycanohydrolase.

Herein, a pectin methyl esterase (EC 3.1.1.11) is any enzyme which iscapable of catalyzing the reaction: pectin+n H₂O→n methanol+pectate. Theenzyme may also been known as pectinesterase, pectin demethoxylase,pectin methoxylase, pectin methylesterase, pectase, pectinoesterase orpectin pectylhydrolase.

Herein, an endo-galactanase (EC 3.2.1.89) is any enzyme capable ofcatalyzing the endohydrolysis of 1,4-β-D-galactosidic linkages inarabinogalactans. The enzyme may also be known as arabinogalactanendo-1,4-β-galactosidase, endo-1,4-β-galactanase, galactanase,arabinogalactanase or arabinogalactan 4-β-D-galactanohydrolase.

Herein, a pectin acetyl esterase is defined herein as any enzyme whichhas an acetyl esterase activity which catalyzes the deacetylation of theacetyl groups at the hydroxyl groups of GaIUA residues of pectin

Herein, an endo-pectin lyase (EC 4.2.2.10) is any enzyme capable ofcatalyzing the eliminative cleavage of (1→4)-α-D-galacturonan methylester to give oligosaccharides with4-deoxy-β-O-methyl-α-D-galact-4-enuronosyl groups at their non-reducingends. The enzyme may also be known as pectin lyase, pectintrans-eliminase; endo-pectin lyase, polymethylgalacturonictranseliminase, pectin methyltranseliminase, pectolyase, PL, PNL or PMGLor (1→4)-6-O-methyl-α-D-galacturonan lyase.

Herein, a pectate lyase (EC 4.2.2.2) is any enzyme capable of catalyzingthe eliminative cleavage of (1→4)-α-D-galacturonan to giveoligosaccharides with 4-deoxy-α-D-galact-4-enuronosyl groups at theirnon-reducing ends. The enzyme may also be known polygalacturonictranseliminase, pectic acid transeliminase, polygalacturonate lyase,endopectin methyltranseliminase, pectate transeliminase,endogalacturonate transeliminase, pectic acid lyase, pectic lyase,α-1,4-D-endopolygalacturonic acid lyase, PGA lyase, PPase-N,endo-α-1,4-polygalacturonic acid lyase, polygalacturonic acid lyase,pectin trans-eliminase, polygalacturonic acid trans-eliminase or(1→4)-α-D-galacturonan lyase.

Herein, an alpha rhamnosidase (EC 3.2.1.40) is any polypeptide which iscapable of catalyzing the hydrolysis of terminal non-reducingα-L-rhamnose residues in α-L-rhamnosides or alternatively inrhamnogalacturonan. This enzyme may also be known as α-L-rhamnosidase T,α-L-rhamnosidase N or α-L-rhamnoside rhamnohydrolase.

Herein, exo-galacturonase (EC 3.2.1.82) is any polypeptide capable ofhydrolysis of pectic acid from the non-reducing end, releasingdigalacturonate. The enzyme may also be known asexo-poly-α-galacturonosidase, exopolygalacturonosidase orexopolygalacturanosidase.

Herein, exo-galacturonase (EC 3.2.1.67) is any polypeptide capable ofcatalyzing:(1,4-α-D-galacturonide)_(n)+H₂O→(1,4-α-D-galacturonide)_(n-1)+D-galacturonate.The enzyme may also be known as galacturan 1,4-α-galacturonidase,exopolygalacturonase, poly(galacturonate) hydrolase,exo-D-galacturonase, exo-D-galacturonanase, exopoly-D-galacturonase orpoly(1,4-α-D-galacturonide) galacturonohydrolase.

Herein, exopolygalacturonate lyase (EC 4.2.2.9) is any polypeptidecapable of catalyzing eliminative cleavage of4-(4-deoxy-α-D-galact-4-enuronosyl)-D-galacturonate from the reducingend of pectate, i.e. de-esterified pectin. This enzyme may be known aspectate disaccharide-lyase, pectate exo-lyase, exopectic acidtranseliminase, exopectate lyase, exopolygalacturonicacid-trans-eliminase, PATE, exo-PATE, exo-PGL or (1→4)-α-D-galacturonanreducing-end-disaccharide-lyase.

Herein, rhamnogalacturonan hydrolase is any polypeptide which is capableof hydrolyzing the linkage between galactosyluronic acid acid andrhamnopyranosyl in an endo-fashion in strictly alternatingrhamnogalacturonan structures, consisting of the disaccharide[(1,2-alpha-L-rhamnoyl-(1,4)-alpha-galactosyluronic acid].

Herein, rhamnogalacturonan lyase is any polypeptide which is anypolypeptide which is capable of cleaving α-L-Rhap-(1→4)-α-D-GalpAlinkages in an endo-fashion in rhamnogalacturonan by beta-elimination.

Herein, rhamnogalacturonan acetyl esterase is any polypeptide whichcatalyzes the deacetylation of the backbone of alternating rhamnose andgalacturonic acid residues in rhamnogalacturonan.

Herein, rhamnogalacturonan galacturonohydrolase is any polypeptide whichis capable of hydrolyzing galacturonic acid from the non-reducing end ofstrictly alternating rhamnogalacturonan structures in an exo-fashion.

Herein, xylogalacturonase is any polypeptide which acts onxylogalacturonan by cleaving the β-xylose substituted galacturonic acidbackbone in an endo-manner. This enzyme may also be known asxylogalacturonan hydrolase.

An α-L-arabinofuranosidase may accept as its substrate bothhemicellulose and pectine, therefore it is classified as both ahemicellulase and a pectinase.

Herein, an α-L-arabinofuranosidase (EC 3.2.1.55) is any polypeptidewhich is capable of acting on α-L-arabinofuranosides, α-L-arabinanscontaining (1,2) and/or (1,3)- and/or (1,5)-linkages, arabinoxylans andarabinogalactans. This enzyme may also be referred to asα-N-arabinofuranosidase, arabinofuranosidase or arabinosidase.

Herein, endo-arabinanase (EC 3.2.1.99) is any polypeptide which iscapable of catalyzing endohydrolysis of 1,5-α-arabinofuranosidiclinkages in 1,5-arabinans. The enzyme may also be know asendo-arabinase, arabinan endo-1,5-α-L-arabinosidase,endo-1,5-α-L-arabinanase, endo-α-1,5-arabanase; endo-arabanase or1,5-α-L-arabinan 1,5-α-L-arabinanohydrolase.

In a special embodiment of the invention, the at least four differentenzymes are at least two different cellobiohydrolases, for example acellobiohydrolase I and/or a cellobiohydrolase II, optionally at leastan endoglucanase, for example β-gluconase CEA and at least aβ-glucosidase.

Preferably, one of said different cellobiohydrolases has at least 70%identity with SEQ ID No. 1 and the other of said differentcellobiohydrolases has at least 70% identity with SEQ ID No. 7, whereinthe endoglucanase has at least 70% identity with SEQ ID No. 3 andwherein the β-glucosidase has at least 70% identity with SEQ ID No. 5.

For example, one cellobiohydrolase may have at least 75%, for example atleast 80%, for example at least 85%, for example at least 90%, forexample at least 93%, for example at least 95%, for example at least97%, for example at least 98%, for example at least 99% identity withSEQ ID NO. 1.

For example, the other cellobiohydrolase may have at least 75%, forexample at least 80%, for example at least 85%, for example at least90%, for example at least 93%, for example at least 95%, for example atleast 97%, for example at least 98%, for example at least 99% identitywith SEQ ID NO. 7.

For example, the endoglucanase may have at least 75%, for example atleast 80%, for example at least 85%, for example at least 90%, forexample at least 93%, for example at least 95%, for example at least97%, for example at least 98%, for example at least 99% identity withSEQ ID NO. 3.

For example, the β-glucosidase may have at least 75%, for example atleast 80%, for example at least 85%, for example at least 90%, forexample at least 93%, for example at least 95%, for example at least97%, for example at least 98%, for example at least 99% identity withSEQ ID NO. 5.

Preferably, the enzymes produced by the host cell of the invention havea cellulase activity of at least 2 WSU in 16 times or more dilutedsupernatant or broth, for example a cellulase activity of at least 3 WSUor a cellulase activity of at least 5 WSU or more in 16 times dilutedsupernatant or broth. The broth or supernatant may for example be 10000times diluted, 5000 times diluted or 2500 times diluted.

With 1 WSU is meant 0.119 mg/ml glucose released from 2.1 w/v % washedpre-treated wheat straw by 200 μl of enzyme mix in 20 hours at 65° C. atpH 4.50.

When the heterologous cellulase, hemicellulase and/or pectinase is to besecreted from the host cell into the cultivation medium, an appropriatesignal sequence can be added to the polypeptide in order to direct thede novo synthesized polypeptide to the secretion route of the host cell.The person skilled in the art knows to select an appropriate signalsequence for a specific host. The signal sequence may be native to thehost cell, or may be foreign to the host cell. As an example, a signalsequence from a protein native to the host cell can be used. Preferably,said native protein is a highly secreted protein, i.e. a protein that issecreted in amounts higher than 10% of the total amount of protein beingsecreted.

As an alternative for a signal sequence, the polypeptide of theinvention can be fused to a secreted carrier protein, or part thereof.Such chimeric construct is directed to the secretion route by means ofthe signal sequence of the carrier protein, or part thereof. Inaddition, the carrier protein will provide a stabilizing effect to thepolypeptide according to the invention and or may enhance solubility.Such carrier protein may be any protein. Preferably, a highly secretedprotein is used as a carrier protein. The carrier protein may be nativeor foreign to the polypeptide according to the invention. The carrierprotein may be native of may be foreign to the host cell. Examples ofsuch carrier proteins are glucoamylase, prepro sequence of alpha-Matingfactor, cellulose binding domain of Clostridium cellulovorans cellulosebinding protein A, glutathione S-transferase, chitin binding domain ofBacillus circulans chitinase A1, maltose binding domain encoded by themalE gene of E. coli K12, beta-galactosidase, and alkaline phosphatase.A preferred carrier protein for expression of such chimeric construct inAspergillus cells is glucoamylase.

The Polynucleotides

The term “polynucleotide” is identical to the term “nucleic acidmolecule” and can herein be read interchangeably. The term refers to apolynucleotide molecule, which is a ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) molecule, either single stranded or doublestranded. A polynucleotide may either be present in isolated form, or becomprised in recombinant nucleic acid molecules or vectors, or becomprised in a host cell. The polynucleotide may be synthetic or may beisolated from chromosomal DNA.

As used herein, the term “gene” refers to nucleic acid molecules, whichmay be isolated from chromosomal DNA or may be synthesized based on asequence listing, which includes an open reading frame encoding apolypeptide.

The polynucleotide according to the invention may be a syntheticpolynucleotide. The synthetic polynucleotide may be optimized in itscodon use, preferably according to the methods described inWO2006/077258 and/or PCT/EP2007/055943, which are herein incorporated byreference. PCT/EP2007/055943 addresses codon-pair optimization.Codon-pair optimisation is a method wherein the nucleotide sequencesencoding a polypeptide have been modified with respect to theircodon-usage, in particular the codon-pairs that are used, to obtainimproved expression of the nucleotide sequence encoding the polypeptideand/or improved production of the encoded polypeptide. Codon pairs aredefined as a set of two subsequent triplets (codons) in a codingsequence.

The term “coding sequence” as defined herein is a sequence, which istranscribed into mRNA and translated into a polypeptide according to theinvention. The boundaries of the coding sequence are generallydetermined by the ATG or other start codon at the 5′-end of the mRNA anda translation stop codon sequence terminating the open reading frame atthe 3′-end of the mRNA. A coding sequence can include, but is notlimited to, DNA, cDNA, and recombinant nucleic acid sequences.

The at least four different heterologous polynucleotides encoding theenzymes chosen from the group of cellulases, hemicellulases andpectinases can be chosen from any of the known polynucleotides.

Preferably, the polynucleotide is chosen from the group ofpolynucleotides encoding cellulases, hemicellulases and pectinases, ischosen from the group of polynucleotides encoding cellulases andhemicellulases, more preferably from the group of polynucleotidesencoding cellulases.

In an embodiment, a polynucleotide according to the invention is chosenfrom the polynucleotides that encode a thermostable enzyme. Herein, thismeans that the enzyme has a temperature optimum of about 60° C. orhigher, for example about 70° C. or higher, such as about 75° C. orhigher, for example about 80° C. or higher such as 85° C. or higher. Theskilled person may select suitable polynucleotides using commonknowledge. They may for example be isolated from thermophilicmicroorganisms, or may be designed by the skilled person andartificially synthesized. In one embodiment the polynucleotides may beisolated from thermophilic filamentous fungi. Examples of thermophilicfilamentous fungi from which polynucleotides encoding thermostableenzymes may be isolated are: Acremonium, Aureobasidium, Cryptococcus,Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora,Neurospora, Paecilomyces, Penicillium, Schizophyllum, Talaromyces,Thermoascus, Thielavia, and Tolypocladium.

In a special aspect of the invention the at least four differentpolynucleotides are at least the polynucleotides encoding acellobiohydrolase I and/or a cellobiohydrolase II, an endoglucanase anda β-glucosidase.

Preferably, the polynucleotide encoding the cellobiohydrolase I has atleast 70%, more preferably at least 75%, more preferably at least 80%,more preferably at least 85%, more preferably at least 87%, for exampleat least 90%, for example at least 93%, for example at least 95%, forexample at least 96%, for example at least 97%, for example at least98%, for example at least 99% or 100% % identity with SEQ ID No. 2(polynucleotide encoding cellobiohydrolase I from Talaromyces emersoniiCBS 39364).

Preferably, the polynucleotide encoding the cellobiohydrolase II has atleast at least 70%, more preferably at least 75%, more preferably atleast 80%, more preferably at least 85%, more preferably at least 87%,for example at least 90%, for example at least 93%, for example at least95%, for example at least 96%, for example at least 97%, for example atleast 98%, for example at least 99% or 100% % identity with SEQ ID No. 8(polynucleotide encoding cellobiohydrolase II from Talaromyces emersoniiCBS 39364).

Preferably, the polynucleotide encoding the endoglucanase has at leastat least 70%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, more preferably at least 87%, forexample at least 90%, for example at least 93%, for example at least95%, for example at least 96%, for example at least 97%, for example atleast 98%, for example at least 99% or 100% % identity with SEQ ID No. 4(polynucleotide encoding endoglucanase from Talaromyces emersonii CBS39364).

Preferably, the polynucleotide encoding the β-glucosidase has at leastat least 70%, more preferably at least 75%, more preferably at least80%, more preferably at least 85%, more preferably at least 87%, forexample at least 90%, for example at least 93%, for example at least95%, for example at least 96%, for example at least 97%, for example atleast 98%, for example at least 99% or 100% % identity with SEQ ID No. 6(polynucleotide encoding 3-glucosidase from Talaromyces emersonii).

In a special embodiment of the invention, the polynucleotide encodingcellobiohydrolase I having at least 70% identity with SEQ ID No. 2(polynucleotide encoding cellobiohydrolase I from Talaromyces emersoniiCBS 39364), the polynucleotide having at least 70% identity with SEQ IDNo. 8 (polynucleotide encoding cellobiohydrolase II from Talaromycesemersonii CBS 39364), the polynucleotide encoding having at least 70%identity with SEQ ID No. 4 (the polynucleotide encoding endoglucanasefrom Talaromyces emersonii CBS 39364) and the polynucleotide having atleast 70% identity with SEQ ID No. 6 (the polynucleotide encodingβ-glucosidase from Talaromyces emersonii) are present in the host cell,preferably an Aspergillus host cell.

Aspergillus is a very good host cell for the polynucleotides ofTalaromyces emersonii: cellobiohydrolase I of SEQ ID No. 2,cellobiohydrolase II of SEQ ID No. 8, endogluconase of SEQ ID No. 4 andβ-glucosidase of SEQ ID No. 6) and those polynucleotides having at least70% identity therewith. Surprisingly, it has been found thatcellobiohydrolase I of SEQ ID No. 2, cellobiohydrolase II of SEQ ID No.8, endogluconase of SEQ ID No. 4 and β-glucosidase of SEQ ID No. 6) areexpressed in and secreted by Aspergillus without any adaptation of thesesequences to the preferred codon usage and signal sequences ofAspergillus.

Preferably, the polynucleotides encoding the at least four differentenzymes chosen from the group of cellulases, hemicellulases andpectinases are original or codon optimized polynucleotides originatingfrom a Talaromyces strain, more preferably from a Talaromyces emersoniistrain, more preferably from the Talaromyces emersonii strain depositedat CENTRAAL BUREAU VOOR SCHIMMELCULTURES, Uppsalalaan 8, P.O. Box 85167,NL-3508 AD Utrecht, The Netherlands on 1 Jul. 2009 having the AccessionNumber CBS 39364, or a functionally equivalent mutant thereof. Thislatter strain is indicated herein as Talaromyces emersonii CBS 39364.

Amino acid or nucleotide sequences are said to be homologous whenexhibiting a certain level of similarity. Whether two homologoussequences are closely related or more distantly related is indicated by“percent identity” or “% identity”, which is high or low respectively.

For the purpose of this invention, it is defined here that in order todetermine the percent identity of two amino acid sequences or of twonucleic acid sequences, the complete sequences are aligned for optimalcomparison purposes. In order to optimize the alignment between the twosequences gaps may be introduced in any of the two sequences that arecompared. Such alignment is carried out over the full length of thesequences being compared. The identity is the percentage of identicalmatches between the two sequences over the reported aligned region.

A comparison of sequences and determination of percent identity betweentwo sequences can be accomplished using a mathematical algorithm. Theskilled person will be aware of the fact that several different computerprograms are available to align two sequences and determine the homologybetween two sequences (Kruskal, J. B. (1983) An overview of sequencecomparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, stringedits and macromolecules: the theory and practice of sequencecomparison, pp. 1-44 Addison Wesley). The percent identity between twoamino acid sequences can be determined using the Needleman and Wunschalgorithm for the alignment of two sequences. (Needleman, S. B. andWunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). The algorithm alignsamino acid sequences as well as nucleotide sequences. TheNeedleman-Wunsch algorithm has been implemented in the computer programNEEDLE. For the purpose of this invention the NEEDLE program from theEMBOSS package was used (version 2.8.0 or higher, EMBOSS: The EuropeanMolecular Biology Open Software Suite (2000) Rice, P. Longden, I. andBleasby, A. Trends in Genetics 16, (6) pp 276-277,http://emboss.bioinformatics.nl/). For protein sequences, EBLOSUM62 isused for the substitution matrix. For nucleotide sequences, EDNAFULL isused. Other matrices can be specified. For purpose of the invention, theparameters used for alignment of amino acid sequences are a gap-openpenalty of 10 and a gap extension penalty of 0.5. The skilled personwill appreciate that all these different parameters will yield slightlydifferent results but that the overall percentage identity of twosequences is not significantly altered when using different algorithms.

The protein sequences mentioned herein can further be used as a “querysequence” to perform a search against sequence databases, for example toidentify other family members or related sequences. Such searches can beperformed using the BLAST programs. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov). BLASTP is usedfor amino acid sequences and BLASTN for nucleotide sequences. In theBLAST program, the default settings may be used:

Cost to open gap: default=5 for nucleotides/11 for proteins

Cost to extend gap: default=2 for nucleotides/1 for proteins

Penalty for nucleotide mismatch: default=−3

Reward for nucleotide match: default=1

Expect value: default=10

Wordsize: default=11 for nucleotides/28 for megablast/3 for proteins

The nucleic acid sequences as mentioned herein can further be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to the nucleicacid molecules of the invention.

Homologous polynucleotide sequences can be isolated, for example, byperforming PCR using two degenerate oligonucleotide primer poolsdesigned on the basis of nucleotide sequences as mentioned herein.

The template for the reaction can be cDNA obtained by reversetranscription of mRNA prepared from strains known or suspected toexpress a polynucleotide according to the invention. The PCR product canbe subcloned and sequenced to ensure that the amplified sequencesrepresent the sequences of a new nucleic acid sequence, or a functionalequivalent thereof.

The PCR fragment can then be used to isolate a full-length cDNA clone bya variety of known methods. For example, the amplified fragment can belabeled and used to screen a bacteriophage or cosmid cDNA library.Alternatively, the labeled fragment can be used to screen a genomiclibrary.

PCR technology also can be used to isolate full-length cDNA sequencesfrom other organisms. For example, RNA can be isolated, followingstandard procedures, from an appropriate cellular or tissue source. Areverse transcription reaction can be performed on the RNA using anoligonucleotide primer specific for the most 5′ end of the amplifiedfragment for the priming of first strand synthesis.

The resulting RNA/DNA hybrid can then be “tailed” (e.g., with guanines)using a standard terminal transferase reaction, the hybrid can bedigested with RNase H, and second strand synthesis can then be primed(e.g., with a poly-C primer). Thus, cDNA sequences upstream of theamplified fragment can easily be isolated. For a review of usefulcloning strategies, see e.g., Sambrook et al., infra.

For example, with the term ‘at least 70% identity with a sequencelisting of an amino acid sequence is meant that the percentage obtainedfrom following the method described above, the percentage obtained is70% or higher.

Process for the Preparation of the Host Cell

In another aspect, the invention relates to a process for thepreparation of a host cell according to the invention comprising thesteps of:

-   -   (a) Providing one or more expression cassettes comprising at        least four different heterologous polynucleotides chosen from        the group of polynucleotides encoding cellulases, hemicellulases        and pectinases and the control sequences required for expression        of these polynucleotides    -   (b) Providing a selectable marker    -   (c) Transforming cells with the one or more expression cassettes        and the selectable marker from step (b)    -   (d) Selecting the thus formed host cells that produce the at        least four different cellulases, hemicellulases and/or        pectinases encoded by the at least four different heterologeous        polynucleotides.

The term “nucleic acid construct” is herein referred to as a nucleicacid molecule, either single- or double-stranded, which is isolated froma naturally occurring gene or which has been modified to containsegments of nucleic acid which are combined and juxtaposed in a mannerwhich would not otherwise exist in nature. The term nucleic acidconstruct is synonymous with the term “expression cassette” when thenucleic acid construct contains all the control sequences required forexpression of a coding sequence, wherein said control sequences areoperably linked to said coding sequence.

The term “operably linked” is defined herein as a configuration in whicha control sequence is appropriately placed at a position relative to thecoding sequence of the DNA sequence such that the control sequencedirects the production of a polypeptide.

The term “control sequences” is defined herein to include allcomponents, which are necessary or advantageous for the expression ofmRNA and/or a polypeptide, either in vitro or in a host cell. Eachcontrol sequence may be native or foreign to the nucleic acid sequenceencoding the polypeptide. Such control sequences include, but are notlimited to, a leader, Shine-Delgarno sequence, optimal translationinitiation sequences (as described in Kozak, 1991, J. Biol. Chem.266:19867-19870), a polyadenylation sequence, a pro-peptide sequence, apre-pro-peptide sequence, a promoter, a signal sequence, and atranscription terminator. At a minimum, the control sequences include apromoter, and transcriptional and translational stop signals. Controlsequences may be optimized to their specific purpose. Preferredoptimized control sequences used in the present invention are thosedescribed in WO2006/077258, which is herein incorporated by reference.

The control sequences may be provided with linkers for the purpose ofintroducing specific restriction sites facilitating ligation of thecontrol sequences with the coding region of the nucleic acid sequenceencoding a polypeptide.

The control sequence may be an appropriate promoter sequence (promoter).

The control sequence may also be a suitable transcription terminator(terminator) sequence, a sequence recognized by a filamentous fungalcell to terminate transcription. The terminator sequence is operablylinked to the 3′-terminus of the nucleic acid sequence encoding thepolypeptide. Any terminator, which is functional in the cell, may beused in the present invention.

Preferred terminator sequences for filamentous fungal cells are obtainedfrom the polynucleotides encoding A. oryzae TAKA amylase, A. nigerglucoamylase (glaA), A. nidulans anthranilate synthase, A. nigeralpha-glucosidase, trpC and Fusarium oxysporum trypsin-like protease.

The control sequence may also be a suitable leader sequence (leaders), anon-translated region of an mRNA which is important for translation bythe filamentous fungal cell. The leader sequence is operably linked tothe 5′-terminus of the nucleic acid sequence encoding the polypeptide.Any leader sequence, which is functional in the cell, may be used in thepresent invention.

Preferred leaders for filamentous fungal cells are obtained from thepolynucleotides encoding A. oryzae TAKA amylase and A. nidulans triosephosphate isomerase and A. niger glaA and phytase.

Other control sequences may be isolated from the Penicillium IPNS gene,or pcbC gene, the beta tubulin gene. All the control sequences cited inWO 01/21779 are herewith incorporated by reference.

The control sequence may also be a polyadenylation sequence, a sequencewhich is operably linked to the 3′-terminus of the nucleic acid sequenceand which, when transcribed, is recognized by the filamentous fungalcell as a signal to add polyadenosine residues to transcribed mRNA. Anypolyadenylation sequence, which is functional in the cell, may be usedin the present invention.

Preferred polyadenylation sequences for filamentous fungal cells areobtained from the polynucleotides encoding A. oryzae TAKA amylase, A.niger glucoamylase, A. nidulans anthranilate synthase, Fusariumoxysporum trypsin-like protease and A. niger alpha-glucosidase.

The term “promoter” is defined herein as a DNA sequence that binds RNApolymerase and directs the polymerase to the correct downstreamtranscriptional start site of a nucleic acid sequence encoding abiological compound to initiate transcription. RNA polymeraseeffectively catalyzes the assembly of messenger RNA complementary to theappropriate DNA strand of a coding region. The term “promoter” will alsobe understood to include the 5′-non-coding region (between promoter andtranslation start) for translation after transcription into mRNA,cis-acting transcription control elements such as enhancers, and othernucleotide sequences capable of interacting with transcription factors.The promoter may be any appropriate promoter sequence suitable for aeukaryotic or prokaryotic host cell, which shows transcriptionalactivity, including mutant, truncated, and hybrid promoters, and may beobtained from polynucleotides encoding extra-cellular or intracellularpolypeptides either homologous (native) or heterologous (foreign) to thecell. The promoter may be a constitutive or inducible promoter.

Examples of inducible promoters that can be used are a starch-, copper-,oleic acid-inducible promoters. The promoter may be selected from thegroup, which includes but is not limited to promoters obtained from thepolynucleotides encoding A. oryzae TAKA amylase, Rhizomucor mieheiaspartic proteinase, A. niger neutral alpha-amylase, A. niger acidstable alpha-amylase, A. niger or A. awamori glucoamylase (glaA), R.miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphateisomerase, A. nidulans acetamidase, the NA2-tpi promoter (a hybrid ofthe promoters from the polynucleotides encoding A. niger neutralalpha-amylase and A. oryzae triose phosphate isomerase), and mutant,truncated, and hybrid promoters thereof.

Preferably, the expression of the at least four polynucleotides encodingcellulase, hemicellulase and/or pectinase is driven by a promoter thatis active in glucose containing medium, for example a glucoamylasepromoter, preferably the glaA promoter as this makes that the presenceof a cellulase inducer is not required.

Therefore, the invention also relates to a host cell capable ofproducing cellulase in the absence of cellulase inducer in a glucosemedium.

Other preferred promoters are the promoters described in WO2006/092396and WO2005/100573, which are herein incorporated by reference.

In order to facilitate expression and/or translation, the polynucleotideor the nucleic acid construct according to the invention may becomprised in an expression vector such that the polynucleotide(s)encoding the enzyme(s) of interest is operably linked to the appropriatecontrol sequences for expression and/or translation in vitro, or inprokaryotic or eukaryotic host cells.

The expression vector may be any vector (e.g., a plasmid or virus),which can be conveniently subjected to recombinant DNA procedures andcan bring about the expression of the nucleic acid sequence encoding thepolypeptide(s) of interest. The choice of the vector will typicallydepend on the compatibility of the vector with the cell into which thevector is to be introduced. The vectors may be linear or closed circularplasmids. The vector may be an autonomously replicating vector, i. e., avector, which exists as an extra-chromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g., a plasmid, anextra-chromosomal element, a mini-chromosome, or an artificialchromosome. An autonomously maintained cloning vector may comprise theAMA1-sequence (see e.g. Aleksenko and Clutterbuck (1997), Fungal Genet.Biol. 21: 373-397).

Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. The integrativecloning vector may integrate at random or at a predetermined targetlocus in the chromosomes of the host cell. In a preferred embodiment ofthe invention, the integrative cloning vector comprises a DNA fragment,which is homologous to a DNA sequence in a predetermined target locus inthe genome of host cell for targeting the integration of the cloningvector to this predetermined locus. In order to promote targetedintegration, the cloning vector is preferably linearized prior totransformation of the cell. Linearization is preferably performed suchthat at least one but preferably either end of the cloning vector isflanked by sequences homologous to the target locus. The length of thehomologous sequences flanking the target locus is preferably at least 30bp, preferably at least 50 bp, preferably at least 0.1 kb, evenpreferably at least 0.2 kb, more preferably at least 0.5 kb, even morepreferably at least 1 kb, most preferably at least 2 kb. Preferably, theefficiency of targeted integration into the genome of the host cell,i.e. integration in a predetermined target locus, is increased byaugmented homologous recombination abilities of the host cell.

Preferably, the homologous flanking DNA sequences in the cloning vector,which are homologous to the target locus, are derived from a highlyexpressed locus meaning that they are derived from a gene, which iscapable of high expression level in the host cell. A gene capable ofhigh expression level, i.e. a highly expressed gene, is herein definedas a gene whose mRNA can make up at least 0.5% (w/w) of the totalcellular mRNA, e.g. under induced conditions, or alternatively, a genewhose gene product can make up at least 1% (w/w) of the total cellularprotein, or, in case of a secreted gene product, can be secreted to alevel of at least 0.1 g/l (as described in EP 357 127 B1).

More than one copy of a nucleic acid sequence may be inserted into thecell to increase production of the product encoded by said sequence.This can be done, preferably by integrating into its genome copies ofthe DNA sequence, more preferably by targeting the integration of theDNA sequence at one of the highly expressed locus defined in the formerparagraph. Alternatively, this can be done by including an amplifiableselectable marker gene with the nucleic acid sequence where cellscontaining amplified copies of the selectable marker gene, and therebyadditional copies of the nucleic acid sequence, can be selected for bycultivating the cells in the presence of the appropriate selectableagent. To increase even more the number of copies of the DNA sequence tobe over expressed the technique of gene conversion as described inWO98/46772 may be used.

The vector system may be a single vector or plasmid or two or morevectors or plasmids, which together contain the total DNA to beintroduced into the genome of the host cell, or a transposon.

The vectors preferably contain one or more selectable markers, whichpermit easy selection of transformed cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like. Theselectable marker may be introduced into the cell on the expressionvector as the expression cassette or may be introduced on a separateexpression vector.

A selectable marker for use in a filamentous fungal cell may be selectedfrom the group including, but not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar(phosphinothricinacetyltransferase), bleA (phleomycin binding), hygB(hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents from otherspecies. Preferred for use in an Aspergillus and Penicillium cell arethe amdS (see for example EP 635574 B1, EP0758020A2, EP1799821A2, WO97/06261A2) and pyrG genes of A. nidulans or A. oryzae and the bar geneof Streptomyces hygroscopicus. More preferably an amdS gene is used,even more preferably an amdS gene from A. nidulans or A. niger. A mostpreferred selectable marker gene is the A. nidulans amdS coding sequencefused to the A. nidulans gpdA promoter (see EP 635574 B1). Otherpreferred AmdS markers are those described in WO2006/040358. AmdS genesfrom other filamentous fungi may also be used (WO 97/06261).

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g. Sambrook & Russell, MolecularCloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor,N.Y., 2001; and Ausubel et al., Current Protocols in Molecular Biology,Wiley InterScience, NY, 1995).

Furthermore, standard molecular cloning techniques such as DNAisolation, gel electrophoresis, enzymatic restriction modifications ofnucleic acids, Southern analyses, transformation of cells, etc., areknown to the skilled person and are for example described by Sambrook etal. (1989) “Molecular Cloning: a laboratory manual”, Cold Spring HarborLaboratories, Cold Spring Harbor, N.Y. and Innis et al. (1990) “PCRprotocols, a guide to methods and applications” Academic Press, SanDiego.

Using the desired polynucleotide sequence as a hybridization probe,nucleic acid molecules used in the invention can be isolated usingstandard hybridization and cloning techniques (e. g., as described inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

A nucleic acid may be amplified using cDNA, mRNA or alternatively,genomic DNA, as a template and appropriate oligonucleotide primersaccording to standard PCR amplification techniques. The nucleic acid soamplified can be cloned into an appropriate vector and characterized byDNA sequence analysis.

Furthermore, oligonucleotides corresponding to or hybridizable to anucleotide sequence according to the invention can be prepared bystandard synthetic techniques, e.g., using an automated DNA synthesizer.

Preferably, the host cell, for example Aspergillus is engineered toimprove the expression of the polynucleotides of interest.

Preferably, the efficiency of targeted integration into the genome ofthe host cell, i.e. integration in a predetermined target locus, isincreased by augmented homologous recombination abilities of the hostcell. Such phenotype of the cell preferably involves a deficient hdfA orhdfB as described in WO2005/095624A2. WO2005/095624A2 discloses apreferred method to obtain a filamentous fungal cell comprisingincreased efficiency of targeted integration.

Optionally, the host cell comprises an elevated unfolded proteinresponse (UPR) compared to the wild type cell to enhance productionabilities of a polypeptide of interest. UPR may be increased bytechniques described in US2004/0186070A1 and/or US2001/0034045A1 and/orWO01/72783A2 and/or WO2005/123763A1. More specifically, the proteinlevel of HAC1 and/or IRE1 and/or PTC2 has been modulated, and/or theSEC61 protein has been engineered in order to obtain a host cell havingan elevated UPR.

Alternatively, or in combination with an elevated UPR, the host cell isgenetically modified to obtain a phenotype displaying lower proteaseexpression and/or protease secretion compared to the wild-type cell inorder to enhance production abilities of a polypeptide of interest. Suchphenotype may be obtained by deletion and/or modification and/orinactivation of a transcriptional regulator of expression of proteases.Such a transcriptional regulator is e.g. prtT. Lowering expression ofproteases by modulation of prtT may be performed by techniques describedin US2004/0191864A1.

Alternatively, or in combination with an elevated UPR and/or a phenotypedisplaying lower protease expression and/or protease secretion, the hostcell displays an oxalate deficient phenotype in order to enhance theyield of production of a polypeptide of interest. An oxalate deficientphenotype may be obtained by techniques described in WO2004/070022A2.

Alternatively, or in combination with an elevated UPR and/or a phenotypedisplaying lower protease expression and/or protease secretion and/oroxalate deficiency, the host cell displays a combination of phenotypicdifferences compared to the wild cell to enhance the yield of productionof the polypeptide of interest. These differences may include, but arenot limited to, lowered expression of glucoamylase and/or neutralalpha-amylase A and/or neutral alpha-amylase B, protease, and oxalicacid hydrolase. Said phenotypic differences displayed by the host cellmay be obtained by genetic modification according to the techniquesdescribed in US2004/0191864A1.

Alternatively, or in combination with an elevated UPR and/or a phenotypedisplaying lower protease expression and/or protease secretion and/oroxalate deficiency and a combination of phenotypic differences comparedto the wild cell to enhance the yield of production of the polypeptideof interest, the host cell displays a deficiency in toxin genes,disabling the ability of the filamentous fungal host cell to expresstoxins. Such toxins include, but are not limited to, ochratoxins,fumonisins, cyclapiazonic acid, 3-nitropropionic acid, emodin,malformin, aflatoxins and secalonic acids. Such deficiency is preferablysuch as described in WO2000/039322A1.

The person skilled in the art knows how to transform cells with the oneor more expression cassettes and the selectable marker. For example, theskilled person may use one or more expression vectors, wherein the oneor more cloning vectors comprise the expression cassettes and theselectable marker.

Transformation of the cells may be conducted by any suitable knownmethods, including e.g. electroporation methods, particle bombardment ormicroprojectile bombardment, protoplast methods and Agrobacteriummediated transformation (AMT). Preferably the protoplast method is used.Procedures for transformation are described by J. R. S. Fincham,Transformation in fungi. 1989, microbiological reviews. 53, 148-170.

Transformation of the host cell by introduction of a polynucleotide anexpression vector or a nucleic acid construct into the cell ispreferably performed by techniques well known in the art (see Sambrook &Russell; Ausubel, supra). Transformation may involve a processconsisting of protoplast formation, transformation of the protoplasts,and regeneration of the cell wall in a manner known per se. Suitableprocedures for transformation of Aspergillus cells are described in EP238 023A2 and Yelton et al., 1984, Proceedings of the National Academyof Sciences USA 81:1470-1474. Suitable procedures for transformation ofAspergillus and other filamentous fungal host cells using Agrobacteriumtumefaciens are described in e.g. De Groot et al., Agrobacteriumtumefaciens-mediated transformation of filamentous fungi. NatBiotechnol. 1998, 16:839-842. Erratum in: Nat Biotechnol 1998 16:1074. Asuitable method of transforming Fusarium species is described byMalardier et al., 1989, Gene 78:147156 or in WO 96/00787A2. Othermethods can be applied such as a method using biolistic transformationas described in: Christiansen et al., Biolistic transformation of theobligate plant pathogenic fungus, Erysiphe graminis fsp. hordei. 1995,Curr Genet. 29:100-102. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

In order to enhance the amount of copies of the polynucleotide ofinterest in the host cell, multiple transformations of the host cell maybe required. In this way, the ratios of the different enzymes producedby the host cell may be influenced. Also, an expression vector maycomprise multiple expression cassettes to increase the amount of copiesof the polynucleotide(s) to be transformed.

Another way could be to choose different control sequences for thedifferent polynucleotides, which—depending on the choice—may cause ahigher or a lower production of the desired enzyme(s).

An advantage of the host cell of the invention is that the host cell ofthe invention can be used for the preparation of an enzyme compositionhaving a consistent composition.

The cells transformed with the selectable marker can be selected basedon the presence of the selectable marker. In case of transformation of(Aspergillus) cells, usually when the cell is transformed with allnucleic acid material at the same time, when the selectable marker ispresent also the polynucleotide(s) encoding the desired enzyme(s) arepresent.

However, in order to ensure that the desired enzymes are produced by thehost cell, the host cell can be selected based on the presence of theenzymes in the fermentation broth. The presence of the desired enzymeactivity (WSU) can be detected in the fermentation broth as described inthe experimental section below. Alternatively, the presence of thedesired enzymes can be determined using SDS-PAGE analysis as describedin the experimental section below.

The Enzyme Composition

In another aspect, the invention relates to the enzyme compositioncomprising at least four different cellulases, hemicellulases and/orpectinases produced by the host cell according to the invention.

The enzyme composition may be the unpurified fermentation broth, whereinthe host cell has produced and secreted the at least four differentcellulases, hemicellulases and/or pectinases, which fermentation brothcontains the host cells or it may be a further purified form of thefermentation broth. Methods for purification of enzymes are known to theperson skilled in the art (downstream processing as described below).

In one embodiment, CBHI is provided in an enzyme composition thatcomprises BG, EG and CBHII. In an embodiment thereof, the amounts ofenzymes are chosen so that BG is present at 2-12%, CBHI at 10-65%, CBHIIat 10-40% and EG at 12-70%, or in an embodiment thereof BG at 4-12%, EGat 18-50%, CBHII at 10-35% and CBHI at 10-60% of the total protein dose(w/w).

A further aspect of the present invention concerns the option ofdownstream processing of the cultivation broth. The batch processapplied according to the invention facilitates downstream processing,especially because of the high yield of the valuable compound and thelow amount of by-products. Downstream processing may include recovery aswell as formulation steps.

After the cultivation process is ended, the valuable product may berecovered from the cultivation broth, using standard technologydeveloped for recovery of the valuable compound of interest. Therelevant downstream processing technology to be applied thereby dependson the nature and cellular localization of the valuable compound and onthe desired purity level of the product of interest. In a typicalrecovery process, the biomass is separated from the cultivation fluidusing e.g. centrifugation or filtration. The valuable compound then isrecovered from the biomass in the case that the valuable product isaccumulated inside or is associated with the microbial cells. The term“recovering” includes isolating, extracting, harvesting, separating orpurifying the compound from culture media. Isolating the compound can beperformed according to any conventional isolation or purificationmethodology known in the art including, but not limited to, alterationof pH, solvent extraction, dialysis, cell filtration andultrafiltration, concentration, lyophilisation and the like.

In yet another aspect, the invention relates to a process for theproduction of a enzyme composition according to the invention comprisingat least four different cellulases, hemicellulases and/or pectinasescomprising the steps of:

-   -   (a) providing the host cell of the invention    -   (b) allowing production and secretion of the at least four        different cellulases, hemicellulases and/or pectinases by the        host cell and    -   (c) optional recovering of the thus obtained enzyme composition.

The invention also relates to the enzyme composition obtainable by thisprocess.

For example, a host cell of the invention may be cultured in a suitablemedium and under conditions allowing the cellulase mixture to beexpressed and/or isolated.

The culture takes place in a suitable nutrient medium comprising carbonand nitrogen sources and inorganic salts, using procedures known in theart (see, e. g., Bennett, J. W. and LaSure, L., eds., More GeneManipulations in Fungi, Academic Press, CA, 1991). Suitable media areavailable from commercial suppliers or may be prepared using publishedcompositions (e. g., in catalogues of the American Type CultureCollection).

The medium, also mentioned herein as cultivation medium, is not criticalto the invention. Nutrients may be added to the process according to theneeds of the host cell in question, provided that the nutrients aresupplied in excess. The cultivation medium conveniently contains acarbon source, a nitrogen source as well as additional compoundsrequired for growth of the microorganism and/or the formation of theproduct. For instance, additional compounds may be present for inducingthe production of the product. Examples of suitable carbon sources knownin the art include glucose, maltose, maltodextrins, sucrose, hydrolysedstarch, starch, molasses and oils. Examples of nitrogen sources known inthe art include soy bean meal, corn steep liquor, yeast extract,ammonia, ammonium salts and nitrate salts. Examples of additionalcompounds include phosphate, sulphate, trace elements and vitamins. Thetotal amount of carbon and nitrogen source to be added may varydepending on e.g. the needs of the host cell and/or the time allowed forexpression and secretion of the enzymes. The ratio between carbon andnitrogen source may vary considerably, whereby one determinant for anoptimal ratio between carbon and nitrogen source may be the elementalcomposition of the product to be formed. Additional compounds requiredfor growth of the host cell and/or for product formation, likephosphate, sulphate or trace elements, may be added in amounts that mayvary between different classes of the host cell, i.e. between fungi,yeasts and bacteria. In addition, the amount of additional compound tobe added may be determined by the type of product formed.

With ‘recovering of the thus obtained enzyme composition’ is meant thatthe enzyme composition may be separated from the host cell and othercomponents that are not the desired enzymes (see downstream processingas described above).

Preferably, the host cell is allowed to produce and secrete the at leastfour different cellulases, hemicellulases and/or pectinases in a glucosecontaining medium, which glucose containing medium preferably does notcontain a cellulase inducer for reasons as described herein.

Of course, the enzyme composition of the present invention may alsocomprise further components, for example proteins, such as enzymes; orother components.

In case of proteins, these proteins may have been produced and secretedby the host cell, but they may have also been separately added to theenzyme composition.

For example, the enzyme composition may comprise an auxiliary enzymeactivity. Such additional activities may be derived from classicalsources and/or produced by a genetically modified organism.

Therefore, in addition, one or more (for example two, three, four orall) enzymes chosen from the group of amylases, proteases, preferablyfrom the group of proteases that do not degrade the cellulases,hemicellulases and/or pectinases present in the enzyme composition;lipases, ligninases, hexosyltransferases, glucuronidases, expansins,cellulose induced proteins, cellulose integrating proteins and otherproteins may be present in the enzyme composition of the invention.

“Protease” includes enzymes that hydrolyze peptide bonds (peptidases),as well as enzymes that hydrolyze bonds between peptides and othermoieties, such as sugars (glycopeptidases). Many proteases arecharacterized under EC 3.4, and are suitable for use in the inventionincorporated herein by reference. Some specific types of proteasesinclude, cysteine proteases including pepsin, papain and serineproteases including chymotrypsins, carboxypeptidases andmetalloendopeptidases.

“Lipase” includes enzymes that hydrolyze lipids, fatty acids, andacylglycerides, including phospoglycerides, lipoproteins,diacylglycerols, and the like. In plants, lipids are used as structuralcomponents to limit water loss and pathogen infection. These lipidsinclude waxes derived from fatty acids, as well as cutin and suberin.

“Ligninase” includes enzymes that can hydrolyze or break down thestructure of lignin polymers. Enzymes that can break down lignin includelignin peroxidases, manganese peroxidases, laccases and feruloylesterases, and other enzymes described in the art known to depolymerizeor otherwise break lignin polymers. Also included are enzymes capable ofhydrolyzing bonds formed between hemicellulosic sugars (notablyarabinose) and lignin. Ligninases include but are not limited to thefollowing group of enzymes: lignin peroxidases (EC 1.11.1.14), manganeseperoxidases (EC 1.11.1.13), laccases (EC 1.10.3.2) and feruloylesterases (EC 3.1.1.73).

“Hexosyltransferase” (2.4.1-) includes enzymes which are capable ofcatalyzing a transferase reaction, but which can also catalyze ahydrolysis reaction, for example of cellulose and/or cellulosedegradation products. An example of a hexosyltransferase which may beused in the invention is a β-glucanosyltransferase. Such an enzyme maybe able to catalyze degradation of (1,3)(1,4)glucan and/or celluloseand/or a cellulose degradation product.

“Glucuronidase” includes enzymes that catalyze the hydrolysis of aglucoronoside, for example 3-glucuronoside to yield an alcohol. Manyglucuronidases have been characterized and may be suitable for use inthe invention, for example β-glucuronidase (EC 3.2.1.31),hyalurono-glucuronidase (EC 3.2.1.36), glucuronosyl-disulfoglucosamineglucuronidase (3.2.1.56), glycyrrhizinate β-glucuronidase (3.2.1.128) orα-D-glucuronidase (EC 3.2.1.139).

The enzyme composition may comprise an expansin or expansin-likeprotein, such as a swollenin (see Salheimo et al., Eur. J. Biohem. 269,4202-4211, 2002) or a swollenin-like protein.

Expansins are implicated in loosening of the cell wall structure duringplant cell growth. Expansins have been proposed to disrupt hydrogenbonding between cellulose and other cell wall polysaccharides withouthaving hydrolytic activity. In this way, they are thought to allow thesliding of cellulose fibers and enlargement of the cell wall. Swollenin,an expansin-like protein contains an N-terminal Carbohydrate BindingModule Family 1 domain (CBD) and a C-terminal expansin-like domain. Forthe purposes of this invention, an expansin-like protein orswollenin-like protein may comprise one or both of such domains and/ormay disrupt the structure of cell walls (such as disrupting cellulosestructure), optionally without producing detectable amounts of reducingsugars.

A composition of the invention may comprise the polypeptide product of acellulose integrating protein, scaffoldin or a scaffoldin-like protein,for example CipA or CipC from Clostridium thermocellum or Clostridiumcellulolyticum respectively.

Scaffoldins and cellulose integrating proteins are multi-functionalintegrating subunits which may organize cellulolytic subunits into amulti-enzyme complex. This is accomplished by the interaction of twocomplementary classes of domain, i.e. a cohesion domain on scaffoldinand a dockerin domain on each enzymatic unit. The scaffoldin subunitalso bears a cellulose-binding module (CBM) that mediates attachment ofthe cellulosome to its substrate. A scaffoldin or cellulose integratingprotein for the purposes of this invention may comprise one or both ofsuch domains.

A composition of the invention may comprise a cellulose induced proteinor modulating protein, for example as encoded by cip1 or cip2 gene orsimilar genes from Trichoderma reesei/Hypocrea jacorina (see Foreman etal., J. Biol. Chem. 278(34), 31988-31997, 2003). The polypeptideproducts of these genes are bimodular proteins, which contain acellulose binding module and a domain which function or activity can notbe related to known glycosyl hydrolase families. Yet, the presence of acellulose binding module and the coregulation of the expression of thesegenes with cellulases components indicates previously unrecognisedactivities with potential role in biomass degradation.

A composition of the invention may be composed of a member of each ofthe classes of the polypeptides mentioned above, several members of onepolypeptide class, or any combination of these polypeptide classes.

A composition of the invention may be composed of polypeptides, forexample enzymes, from (1) commercial suppliers; (2) cloned genesexpressing polypeptides, for example enzymes; (3) complex broth (such asthat resulting from growth of a microbial strain in media, wherein thestrains secrete proteins and enzymes into the media; (4) cell lysates ofstrains grown as in (3); and/or (5) plant material expressingpolypeptides, for example enzymes. Different polypeptides, for exampleenzymes in a composition of the invention may be obtained from differentsources.

The activities in the enzyme composition may be thermostable. Herein,this means that the activity has a temperature optimum of about 60° C.or higher, for example about 70° C. or higher, such as about 75° C. orhigher, for example about 80° C. or higher such as 85° C. or higher.Activities in the enzyme composition will typically not have the sametemperature optima, but preferably will, nevertheless, be thermostable.

In addition, enzyme activities in the enzyme composition may be able towork at low pH. For the purposes of this invention, low pH indicates apH of about 5.5 or lower, about 5 or lower, about 4.5 or lower, about4.0 or lower or about 3.8 or lower or about 3.5 or lower.

Activities in the enzyme composition may be defined by a combination ofany of the above temperature optima and pH values.

Industrial Application of the Enzyme Composition

In principle, an enzyme composition of the invention may be used in anyprocess which requires the treatment of a material which comprisesnon-starch polysaccharide. Thus, a polypeptide or enzyme composition ofthe invention may be used in the treatment of non-starch polysaccharidematerial. Herein, non-starch polysaccharide material is a material whichcomprises or consists essential of one or, more typically, more than onenon-starch polysaccharide.

Typically, plants and fungi and material derived therefrom comprisesignificant quantities of non-starch polysaccharide material.Accordingly, a polypeptide of the invention may be used in the treatmentof a plant or fungal material or a material derived therefrom.

An important component of plant non-starch polysaccharide material islignocellulose (also referred to herein as lignocellulolytic biomass).Lignocellulose is plant material that is composed of cellulose andhemicellulose and lignin. The carbohydrate polymers (cellulose andhemicelluloses) are tightly bound to the lignin by hydrogen and covalentbonds. Accordingly, a polypeptide of the invention may be used in thetreatment of lignocellulolytic material. Herein, lignocellulolyticmaterial is a material which comprises or consists essential oflignocellulose. Thus, in a method of the invention for the treatment ofa non-starch polysaccharide, the non-starch polysaccharide may be alignocellulosic material/biomass.

Accordingly, the invention provides a method of treating a non-starchpolysaccharide in which the treatment comprises the degradation and/ormodification of cellulose and/or hemicellulose.

Degradation in this context indicates that the treatment results in thegeneration of hydrolysis products of cellulose and/or hemicelluloseand/or a pectic substance, i.e. saccharides of shorter length arepresent as result of the treatment than are present in a similaruntreated non-starch polysaccharide. Thus, degradation in this contextmay result in the liberation of oligosaccharides and/or sugar monomers.

All plants and fungi contain non-starch polysaccharide as do virtuallyall plant- and fungal-derived polysaccharide materials. Accordingly, ina method of the invention for the treatment of a non-starchpolysaccharide, said non-starch polysaccharide may be provided in theform of a plant or a plant derived material or a material comprising aplant or plant derived material, for example a plant pulp, a plantextract, a foodstuff or ingredient therefore, a fabric, a textile or anitem of clothing.

The enzyme compositions of the invention can extremely effectivelyhydrolyze lignocellulolytic material, for example corn stover or wheatstraw, into monomeric sugars (the hydrolysis of lignocellulosic materialis also referred to herein as saccharification of lignocellulosicmaterial) which can then be further converted into a useful product,such as ethanol.

Therefore, in another aspect, the invention relates to a process for thesaccharification of lignocellulosic material comprising the steps of

optional pretreatment of lignocellulosic material and

contacting the lignocellulosic material with a enzyme compositionaccording to the invention to produce one or more sugars.

The enzyme compositions of the invention can be used to carry out highlyeffective hydrolysis of a lignocellulosic substrate. This is highlysignificant in the context of commercially viable fuel ethanolproduction from lignocellulosic biomass since lower amounts of enzymewill be required (as compared with currently available products).

In addition, currently available enzymes having cellulase activity,typically derived from Trichoderma, function at mesophilic temperatures,such as from 45° C. to 50° C. and at pH 5.0. This, however, may lead tobacterial infection reducing product yield, so it is desirable to carryout saccharification at a temperature of 65° C. or higher. In addition,the use of mesophilic temperatures increases the viscosity of thebiomass being used such that the dry matter content used is limited.Also, when acid pretreated biomass is used as a substrate, the pH mustbe raised so that the enzyme can saccharify the sugars in the biomass.In the context of a commercially viable fuel ethanol industry, thisimplies a requirement for, for example, sodium hydroxide or calciumsulphate and the production of huge quantities of the correspondingsalts, for example gypsum in the case of sodium hydroxide. Accordingly,it is desirable to carry out saccharification using an enzyme which canoperate at a pH of pH 4.0 or lower.

Moreover, this hydrolysis may be carried out at a high temperatureespecially, if the polynucleotides encoding the at least four differentenzymes chosen from the group of polynucleotides encoding cellulases,hemicellulases and pectinases are polynucleotides originating from aTalaromyces strain, for example from a Talaromyces emersonii strain.

Hydrolysis of a lignocellulosic substrate at a higher temperature, forexample at a temperature of 65° C. or higher, is favorable as it (i)reduces the risk of bacterial infection and (ii) results in a lessviscous biomass pulp. The effect of the latter is significant since itenables the better blending of enzymes, resulting in a higheroperational dry matter in the plant and allows a consequent higherethanol concentration to be achieved. Thus, less energy need be usedimproving sustainability and a smaller fermentation process will berequired requiring lower investment.

A pretreatment of the lignocellulosic material usually occurs at hightemperature; therefore if the polynucleotides encoding the at least fourdifferent enzymes are chosen from the group of polynucleotides encodingcellulases, hemicellulases or pectinases are polynucleotides originatingfrom a Talaromyces strain, for example from a Talaromyces emersoniistrain, there is no need to completely cool the lignocellulosic materialto low temperatures, such as below 40° C. before the enzyme compositionis added. Instead, the enzyme composition may for example be added tothe lignocellulosic material having a temperature of up to 80° C., forexample to lignocellulosic material having a temperature in the range offrom 60 to 80° C., for instance at about 65° C.

This shortens the process for the saccharification of lignocellulosicmaterial considerably, as well as provides for a greener route. Thereason for this is that addition of enzymes at higher temperaturesrequires less cooling of the pre-treated lignocellulosic materials,which saves energy, and enzymes can be added earlier to thelignocellulosic material, which saves time.

For example, the hydrolysis of the lignocellulosic material by theenzyme composition of the invention may be performed at a temperature ofat least 30° C., for example of at least 37° C., for example of at least40° C., for example of at least 50° C., for example of at least 56° C.,for example of at least 60° C., for example of at least 65° C., forexample of at least 70° C.

For example, the hydrolysis of the lignocellulosic material by theenzyme composition of the invention may be performed at a temperature ofat most 100° C., for example of at most 90° C., for example of at most80° C., for example of at most 70° C.

Also, this hydrolysis may be carried out at low pH, for example at a pHof pH 4.0 or lower. This is desirable since biomass is often pretreatedwith acid. Biomass treated in this way does not have to be pH adjustedif the enzymes subsequently used for saccharification are capable ofacting at low pH. This implies a lower requirement of, for example,sodium hydroxide or calcium sulphate and a process in which there is nowaste salt. This is significant in a process in which, for example, fuelethanol is to be produced since huge quantities of material are consumedin such processes. This allows the process according to the invention tobe carried out with no or little pH adjustment is required, i.e. thereis no or a reduced requirement for the addition of acids or bases. Theprocess may thus be carried out as a zero or low waste process (no orlow salt production) and/or as a process in which no or little inorganicchemical input is required.

In addition, it has been shown that the enzyme composition caneffectively hydrolyze biomass when high dry matter contents are used. Itis highly desirable that enzymes used in the production of, for example,fuel ethanol are able to operate on substrates having high viscosity(i.e. high dry weight composition) since this allows higher amounts ofthe final product, for example, fuel ethanol, to be achieved.

Significantly, a method of the invention may be carried out using highlevels of dry matter (of the lignocellulosic material) in the hydrolysisreaction. Thus, the invention may be carried out with a dry mattercontent of about 5% or higher, about 8% or higher, about 10% or higher,about 15% or higher, about 20% or higher, about 25% or higher, about 30%or higher, about 35% or higher or about 40% or higher.

Preferably in the method, the lignocellulosic material is orchardprimings, chaparral, mill waste, urban wood waste, municipal waste,logging waste, forest thinnings, short-rotation woody crops, industrialwaste, wheat straw, oat straw, rice straw, barley straw, rye straw, flaxstraw, soy hulls, rice hulls, rice straw, corn gluten feed, sugar cane,corn stover, corn stalks, corn cobs, corn husks, miscanthus, sweetsorghum, canola stems, soybean stems, prairie grass, gamagrass, foxtail;sugar beet pulp, citrus fruit pulp, seed hulls, cellulosic animalwastes, lawn clippings, cotton, seaweed, softwood, poplar, pine, shrubs,grasses, wheat, sugar cane bagasse, corn, corn hobs, corn kernel, fiberfrom kernels, products and by-products from wet or dry milling ofgrains, municipal solid waste, waste paper, yard waste, herbaceousmaterial, agricultural residues, forestry residues, waste paper, pulp,paper mill residues, branches, bushes, canes, an energy crop, forest, afruit, a flower, a grain, a grass, a herbaceous crop, a leaf, bark, aneedle, a log, a root, a sapling, a shrub, switch grass, a tree, avegetable, fruit peel, a vine, sugar beet pulp, wheat midlings, oathulls, hard or soft wood, organic waste material generated from anagricultural process, forestry wood waste, or a combination of any twoor more thereof.

The composition is reacted with substrate under any appropriateconditions. For example, enzymes can be incubated at about 25° C., about30° C., about 35° C., about 37° C., about 40° C., about 45° C., about50° C., about 55° C., about 60° C., about 65° C., about 70° C., about75° C., about 80° C., about 85° C., about 90° C. or higher. That is,they can be incubated at a temperature of from about 20° C. to about 95°C., for example in buffers of low to medium ionic strength and/or fromlow to neutral pH. By “medium ionic strength” is intended that thebuffer has an ion concentration of about 200 millimolar (mM) or less forany single ion component. The pH may range from about pH 2.5, about pH3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5, about pH 5.5,about pH 6, about pH 6.5, about pH 7, about pH 7.5, about pH 8.0, toabout pH 8.5. Generally, the pH range will be from about pH 3.0 to aboutpH 9.

Typically, the reaction may be carried out under low pH conditions asdefined above. Thus, a method of the invention may be carried out suchthat no pH adjustment (i.e. to a more neutral pH is required). That isto say, an acid pretreated feedstock may be used as is with norequirement to addition of, for example, sodium hydroxide, prior toaddition of an enzyme composition of the invention.

The feedstock may be washed prior to liquefaction/hydrolysis. Suchwashing may be with, for example, water.

Incubation of a composition under these conditions results in release orliberation of substantial amounts of the sugar from the lignocellulosicmaterial. By substantial amount is intended at least about 20%, at leastabout 30%, at least about 40%, at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95% or more of available sugar.

A liquefaction/hydrolysis or presaccharification step involvingincubation with an enzyme or enzyme mixture can be utilized. This stepcan be performed at many different temperatures but it is preferred thatthe presaccharification occur at the temperature best suited to theenzyme mix being tested, or the predicted enzyme optimum of the enzymesto be tested. The temperature of the pretreatment may range from about10° C. to about 100° C., about 30° C. to about 80° C., about 40° C. toabout 70° C., about 50° C. to about 70° C., preferably about 60° C. toabout 70° C., more preferably about 65° C. In the absence of data on thetemperature optimum, it is preferable to perform the pretreatmentreactions at 37° C. first, then at a higher temperature such as 65° C.The pH of the presaccharification mixture may range from about 2.0 toabout 10.0, but is preferably about 3.0 to about 5.0. Again, it may notbe necessary to adjust the pH prior to saccharification since the enzymecomposition is typically suitable for use at low pH as defined herein.

The liquefaction/hydrolysis or presaccharification step reaction mayoccur from several minutes to several hours, such as from about 1 hourto about 120 hours, preferably from about 2 hours to about 48 hours,more preferably from about 2 to about 24 hours, most preferably for fromabout 2 to about 6 hours. The liquification/hydrolysis may occur fromseveral minutes to several hours, such as from about 6 hours to about168 hours, preferably about 12 hours to about 96 hours, more preferablyabout 24 hours to about 72 hours, even more preferably from about 24hours to about 48 hours.

Depending on the application, e.g. when the enzymes or enzymecomposition are used in a process where liquifaction/hydrolysis iscombined with fermentation, the process may be carried out as separatehydrolysis and fermentation (SHF) and simultaneous hydrolysis andfermentation (SSF). In SSF a presaccharifation may be appropriate. SHFand SSF will be described in more detail below.

The invention further relates to a method for the conversion oflignocellulosic material into useful product comprising the followingsteps: a) pretreatment of one or more lignocellulosic material toproduce pretreated lignocellulosic material; b) enzymatic treatment ofthe pretreated lignocellulosic material to produce one or more sugar; c)converting the sugar into one or more useful product and d) separatingthe one or more useful products, wherein in step a), b) or c) an enzymecomposition of the invention is used or added.

Therefore, the invention also relates to a process for the preparationof a fermentation product, including amino acids, vitamins,pharmaceuticals, animal feed supplements, specialty chemicals, chemicalfeedstocks, plastics, solvents, fuels, or other organic polymers, lacticacid, ethanol, fuel ethanol or chemicals, plastics, dicarboxylic acids,such as for example succinic acid, itaconic acid, adipic acid;(bio)fuels, including ethanol, methanol, butanol, synthetic liquid fuelsand biogas, wherein the one or more sugars that is or are producedaccording to the process of the invention, is fermented with afermenting microorganism, preferably yeast to produce the fermentationproduct.

With ‘fermenting microorganism’ is meant a microorganism having theability to convert the one or more sugars into the fermentation product.

Such a process may be carried out without any requirement to adjust thepH during the process. That is to say, the process is one which may becarried out without the addition of any acid(s) or base(s). However,this excludes a pretreatment step, where acid may be added. The point isthat the enzyme composition of the invention is capable of acting at lowpH and, therefore, there is no need to adjust the pH of acid of an acidpretreated feedstock in order that saccharification may take place.Accordingly, a method of the invention may be a zero waste method usingonly organic products with no requirement for inorganic chemical input.

Preferably, in the method according to the invention, the useful productis one or more of ethanol, butanol, lactic acid, a plastic, an organicacid, a solvent, an animal feed supplement, a pharmaceutical, a vitamin,an amino acid, an enzyme or a chemical feedstock. The present inventionprovides relates to a composition which comprises cellulolytic and/orhemicellulolytic enzyme activity and which has the ability to modify,for example degrade, a non-starch carbohydrate material. A non-starchcarbohydrate material is a material which comprises, consists of orsubstantially consists of one or more non-starch carbohydrates.Carbohydrate in this context includes all saccharides, for examplepolysaccharides, oligosaccharides, disaccharides or monosaccharides.

A composition as described herein typically modifies a non-starchcarbohydrate material by chemically modification of such material.Chemical modification of the carbohydrate material may result in thedegradation of such material, for example by hydrolysis, oxidation orother chemical modification such as by the action of a lyase.

A non-starch carbohydrate suitable for modification by a composition asdescribed herein is lignocellulose. The major polysaccharides comprisingdifferent lignocellulosic residues, which may be considered as apotential renewable feedstock, are cellulose (glucans), hemicelluloses(xylans, heteroxylans and xyloglucans). In addition, some hemicellulosemay be present as glucomannans, for example in wood-derived feedstocks.The enzymatic hydrolysis of these polysaccharides to soluble sugars,including both monomers and multimers, for example glucose, cellobiose,xylose, arabinose, galactose, fructose, mannose, rhamnose, ribose,galacturonic acid, glucoronic acid and other hexoses and pentoses occursunder the action of different enzymes acting in concert.

In addition, pectins and other pectic substances such as arabinans maymake up considerably proportion of the dry mass of typically cell wallsfrom non-woody plant tissues (about a quarter to half of dry mass may bepectins).

Cellulose is a linear polysaccharide composed of glucose residues linkedby β-1,4 bonds. The linear nature of the cellulose fibers, as well asthe stoichiometry of the β-linked glucose (relative to α) generatesstructures more prone to interstrand hydrogen bonding than the highlybranched α-linked structures of starch. Thus, cellulose polymers aregenerally less soluble, and form more tightly bound fibers than thefibers found in starch.

A method for the preparation of a fermentation product may optionallycomprise recovery of the fermentation product.

Such a process may be carried out under aerobic or anaerobic conditions.Preferably, the process is carried out under micro-aerophilic or oxygenlimited conditions.

An anaerobic fermentation process is herein defined as a fermentationprocess run in the absence of oxygen or in which substantially no oxygenis consumed, preferably about 5 or less, about 2.5 or less or about 1mmol/L/h or less, and wherein organic molecules serve as both electrondonor and electron acceptors.

An oxygen-limited fermentation process is a process in which the oxygenconsumption is limited by the oxygen transfer from the gas to theliquid. The degree of oxygen limitation is determined by the amount andcomposition of the ingoing gasflow as well as the actual mixing/masstransfer properties of the fermentation equipment used. Preferably, in aprocess under oxygen-limited conditions, the rate of oxygen consumptionis at least about 5.5, more preferably at least about 6 and even morepreferably at least about 7 mmol/L/h. A method for the preparation of afermentation product may optionally comprise recovery of thefermentation product.

In the absence of oxygen, NADH produced in glycolysis and biomassformation, cannot be oxidised by oxidative phosphorylation. To solvethis problem many microorganisms use pyruvate or one of its derivativesas an electron and hydrogen acceptor thereby regenerating NAD⁺.

Thus, in a preferred anaerobic fermentation process pyruvate is used asan electron (and hydrogen acceptor) and is reduced to fermentationproducts such as ethanol, butanol, lactic acid, 3-hydroxy-propionicacid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid,fumaric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, aβ-lactam antibiotic and a cephalosporin.

The fermentation process is preferably run at a temperature that isoptimal for the cell. Thus, for most yeasts or fungal host cells, thefermentation process is performed at a temperature which is less thanabout 42° C., preferably less than about 38° C. For yeast or filamentousfungal host cells, the fermentation process is preferably performed at atemperature which is lower than about 35, about 33, about 30 or about28° C. and at a temperature which is higher than about 20, about 22, orabout 25° C.

The ethanol yield on xylose and/or glucose in the process preferably isat least about 50, about 60, about 70, about 80, about 90, about 95 orabout 98%. The ethanol yield is herein defined as a percentage of thetheoretical maximum yield.

The invention also relates to a process for producing a fermentationproduct.

In a preferred process the cell ferments both the xylose and glucose,preferably simultaneously in which case preferably a cell is used whichis insensitive to glucose repression to prevent diauxic growth. Inaddition to a source of xylose (and glucose) as carbon source, thefermentation medium will further comprise the appropriate ingredientrequired for growth of the cell. Compositions of fermentation media forgrowth of microorganisms such as yeasts are well known in the art

The fermentation processes may be carried out in batch, fed-batch orcontinuous mode. A separate hydrolysis and fermentation (SHF) process ora simultaneous saccharification and fermentation (SSF) process may alsobe applied. A combination of these fermentation process modes may alsobe possible for optimal productivity. These processes are describedhereafter in more detail.

SSF Mode

For Simultaneous Saccharification and Fermentation (SSF) mode, thereaction time for liquefaction/hydrolysis or presaccharification step isdependent on the time to realize a desired yield, i.e. cellulose toglucose conversion yield. Such yield is preferably as high as possible,preferably 60% or more, 65% or more, 70% or more, 75% or more 80% ormore, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more,98% or more, 99% or more, even 99.5% or more or 99.9% or more.

According to the invention very high sugar concentrations in SHF modeand very high product concentrations (e.g. ethanol) in SSF mode arerealized. In SHF operation the glucose concentration is 25 g/L or more,30 g/L or more, 35 g/L or more, 40 g/L or more, 45 g/L or more, 50 g/Lor more, 55 g/L or more, 60 g/L or more, 65 g/L or more, 70 g/L or more,75 g/L or more, 80 g/L or more, 85 g/L or more, 90 g/L or more, 95 g/Lor more, 100 g/L or more, 110 g/L or more, 120 g/L or more or may e.g.be 25 g/L-250 g/L, 30 g/L-200 g/L, 40 g/L-200 g/L, 50 g/L-200 g/L, 60g/L-200 g/L, 70 g/L-200 g/L, 80 g/L-200 g/L, 90 g/L, 80 g/L-200 g/L.

Product Concentration in SSF Mode

In SSF operation, the product concentration (g/L) is dependent on theamount of glucose produced, but this is not visible since sugars areconverted to product in the SSF, and product concentrations can berelated to underlying glucose concentration by multiplication with thetheoretical maximum yield (Yps max in gr product per gram glucose)

The theoretical maximum yield (Yps max in gr product per gram glucose)of a fermentation product can be derived from textbook biochemistry. Forethanol, 1 mole of glucose (180 gr) yields according to normalglycolysis fermentation pathway in yeast 2 moles of ethanol (=2×46=92 grethanol. The theoretical maximum yield of ethanol on glucose istherefore 92/180=0.511 gr ethanol/gr glucose.

For Butanol (MW 74 gr/mole) or iso butanol, the theoretical maximumyield is 1 mole of butanol per mole of glucose. So Yps max for(iso-)butanol=74/180=0.411 gr (iso-) butanol/gr glucose.

For lactic acid the fermentation yield for homolactic fermentation is 2moles of lactic acid (MW=90 gr/mole) per mole of glucose. According tothis stoichiometry, the Yps max=1 gr lactic acid/gr glucose.

For other fermentation products a similar calculation may be made.

SSF Mode

In SSF operation the product concentration is 25 g*Yps g/L/L or more,30*Yps g/L or more, 35 g*Yps/L or more, 40*Yps g/L or more, 45*Yps g/Lor more, 50*Yps g/L or more, 55*Yps g/L or more, 60*Yps g/L or more,65*Yps g/L or more, 70*Yps g/L or more, 75*Yps g/L or more, 80*Yps g/Lor more, 85*Yps g/L or more, 90*Yps g/L or more, 95*Yps g/L or more,100*Yps g/L or more, 110*Yps g/L or more, 120 g/L*Yps or more or maye.g. be 25*Yps g/L-250*Yps g/L, 30*Yps gl/L-200*Yps g/L, 40*Ypsg/L-200*Yps g/L, 50*Yps g/L-200*Yps g/L, 60*Yps g/L-200*Yps g/L, 70*Ypsg/L-200*Yps g/L, 80*Yps g/L-200*Yps g/L, 90*Yps g/L, 80*Yps g/L-200*Ypsg/L

Accordingly, the invention provides a method for the preparation of afermentation product, which method comprises:

a. degrading lignocellulose using a method as described herein; and

b. fermenting the resulting material,

thereby to prepare a fermentation product.

Fermentation Product

The fermentation product of the invention may be any useful product. Inone embodiment, it is a product selected from the group consisting ofethanol, n-butanol, isobutanol, lactic acid, 3-hydroxy-propionic acid,acrylic acid, acetic acid, succinic acid, fumaric acid, malic acid,itaconic acid, maleic acid, citric acid, adipic acid, an amino acid,such as lysine, methionine, tryptophan, threonine, and aspartic acid,1,3-propane-diol, ethylene, glycerol, a β-lactam antibiotic and acephalosporin, vitamins, pharmaceuticals, animal feed supplements,specialty chemicals, chemical feedstocks, plastics, solvents, fuels,including biofuels and biogas or organic polymers, and an industrialenzyme, such as a protease, a cellulase, an amylase, a glucanase, alactase, a lipase, a lyase, an oxidoreductases, a transferase or axylanase.

Recovery of the Fermentation Product

For the recovery of the fermentation product existing technologies areused. For different fermentation products different recovery processesare appropriate. Existing methods of recovering ethanol from aqueousmixtures commonly use fractionation and adsorption techniques. Forexample, a beer still can be used to process a fermented product, whichcontains ethanol in an aqueous mixture, to produce an enrichedethanol-containing mixture that is then subjected to fractionation(e.g., fractional distillation or other like techniques). Next, thefractions containing the highest concentrations of ethanol can be passedthrough an adsorber to remove most, if not all, of the remaining waterfrom the ethanol.

The invention will now be elucidated by way of the following examples,without however being limited thereto.

Example 1 Expression of Multiple Talaromyces Emersonii Cellulases inAspergillus Niger

This example describes the cloning and expression of T. emersonii CBS39364 cellobiohydrolase-I (CBHI), T. emersonii CBS 39364cellobiohydrolase-II (CBHII), T. emersonii β-glucanase CEA CBS 39364(EG), and T. emersonii β-glucosidase (BG) as presented in the GenBankdatabase with accession number AAL69548 in A. niger WT-1. In addition,cellulase activity of transformants is compared with cellulase activityof an empty strain after growing the strains in shake flasks.

Cloning of T. emersonii Coding Regions in Expression Vectors

The polynucleotides (DNA-sequences) encoding T. emersoniicellobiohydrolase-I (CBHI), T. emersonii beta-glucanase CEA (EG), and T.emersonii β-glucosidase (BG) were synthesised by DNA2.0 (Menlo Park,USA) and cloned as EcoRI/SnaBI fragment into the pGBTOP-8 vector (EP0635574A1), comprising the glucoamylase (glaA) promoter and terminatorsequence, resulting in vector pGBTOPEBA205, pGBTOPEBA8 and pGBTOPEBA4,respectively. For cloning purposes, 198 nucleotides of the 3′ part ofthe glucoamylase promoter was also synthesised with the codingsequences. The amino acid sequences of the T. emersoniicellobiohydrolase-I (CBHI), T. emersonii beta-glucanase CEA (EG), and T.emersonii β-glucosidase (BG) are represented by SEQ ID NO: 1, 3, and 5,respectively. The DNA sequences are represented by SEQ ID NO: 2, 4 and6, respectively. FIG. 1 represents a map of a pGBTOPEBA205 containingthe DNA-sequence encoding the T. emersonii CBHI under control of theglaA promoter within vector pGBTOP12. pGBTOPEBA205 is representative forpGBTOPEBA8, which comprises the DNA-sequence encoding the T. emersoniiEG, and pGBTOPEBA4, which comprises DNA-sequence encoding the T.emersonii BG.

The polynucleotide encoding T. emersonii cellobiohydrolase-II (CBHII),was obtained from a T. emersonii cDNA library as described in patentWO2001/070998 A1. FIG. 2 represents a map of pGBFINEBA176 containing theDNA-sequence encoding the T. emersonii CBHII under control of the glaApromoter within vector pGBFIN11 (WO 9932617 (A2)). The pGBFIN11 vectoralso contains the selection marker, AmdS (EP0758020 (A2), which selectsfor the ability to use acetamide as sole nitrogen source. The amino acidsequence and nucleotide sequence are represented by SEQ ID NO: 7 and 8,respectively.

Transformation of Asperqillus Niger with Multiple Cellulases

Aspergillus niger WT-1 strain was used for transformations. TheAspergillus niger WT-1 strain is derived from the A. niger straindeposited at the CBS Institute under the deposit number CBS 513.88 (Pelet al., Genome sequencing and analysis of the versatile cell factoryAspergillus niger CBS 513.88. 2007, Nat Biotechnol. 25:189-90). Itcomprises a deletion of the gene encoding glucoamylase (glaA), which wasconstructed by using the “MARKER-GENE FREE” approach as described in EP0635574 (A1). In this patent application it is extensively described howto delete glaA specific DNA sequences in the genome of CBS 513.88. Theprocedure resulted in a MARKER-GENE FREE ΔglaA recombinant A. nigerCBS513.88 strain, possessing finally no foreign DNA sequences at all.

In order to introduce the pGBTOPEBA vectors and pGBFINEBA176 inAspergillus niger WT-1, transformation and subsequent transformantselection was carried out essentially as described in WO 9846772 (A2)and WO 9932617 (A2). In brief, linear DNA of the vector was isolatedafter digestion with NotI, to remove the E. coli sequences from thevector. After transformation of the cells with the linear DNA,transformants were selected on media comprising acetamide as solenitrogen source and colony purified.

Transformants were cultured in shake flasks in 100 ml of CSM-MES mediumas described in EP 635 574 A1 at 34° C. at 170 rpm in an incubatorshaker using a 500 ml baffled shake flask. After different time-pointsof fermentation, supernatant samples were harvested to determineexpression by SDS-PAGE analysis. Protein samples were separated underreducing conditions on NuPAGE 4-12% Bis-Tris gel (Invitrogen, Breda, TheNetherlands) and stained with Sypro Ruby (Invitrogen, Breda, TheNetherlands)) according to manufacturer's instructions.

FIG. 3A shows the SDS-PAGE gel of a subset of tested transformantsexpressing multiple cellulases. The theoretical molecular weights of theproteins encoded by pGBTOPEBA4, pGBTOPEBA8, pGBFINEBA176 andpGBTOPEBA205 are shown in Table 1.

TABLE 1 Theoretical molecular weight of the proteins encoded bypGBTOPEBA4, pGBTOPEBA8, pGBFINEBA176 and pGBTOPEBA205 Protein encoded byTheoretical molecular Construct construct weight pGBTOPEBA4 BG 92 kDapGBTOPEBA8 EG 37 kDa pGBFINEBA176 CBHII 48 kDa pGBTOPEBA205 CBHI 49 kDa

As expected, different combinations of overexpressed cellulases wereobserved. From the SDS-PAGE analysis a host cell capable of expressingand secreting the at least four different cellulases, such as forexample transformant 19, can be selected.

To determine whether the transformants were able to degrade pre-treatedwheat straw a WSU assay was performed to determine cellulase activity.

Wheat Straw Assay (WSU Assay)

In order to measure cellulase activity a WSU activity assay wasperformed. WSU activity was measures in supernatants (the liquid part ofthe broth wherein the cells were cultured) of an empty strain and thetransformant:

Preparation of Pre-Treated, Washed Wheat Straw Substrate.

Dilute-acid pre-treated wheat straw which was washed with water untilthe solution with wheat straw was pH 6.5 or higher and the mass washomogenised using an ultra-turrax, lyophilized and grinded prior toanalysis. To obtain pre-treated wheat straw a dilute acid pre-treatmentas described in Linde, M. et al, Biomass and Bioenergy 32 (2008),326-332 and equipment as described in Schell, D. J., AppliedBiochemistry and Biotechnology (2003), vol. 105-108, pp 69-85, may beused.

With 1 WSU is meant 0.119 mg/ml glucose released from 2.1 w/v washedpre-treated wheat straw by 200 μl of enzyme mix in 20 hours at 65° C. atpH 4.50.

The glucose release is not a linear function of the quantity of enzymein the composition. In other words, twice the amount of enzyme does notautomatically result in twice the amount of glucose in equal time.Therefore, it is preferred to choose the dilution of the composition tobe tested for WSU activity such that a WSU does not exceed 40.

Measurement of Cellulase Activity in WSU/ml

400 μl of supernatants harvested from shake flask experiments werediluted 16-fold. Diluted sample was used to perform two measurements inwhich 200 μl of diluted sample was analysed. In the first measurement,200 μl diluted sample was transferred to a vial containing 700 μL watercontaining 3% (w/v) dry matter of the pretreated washed wheat strawsubstrate and 100 μl of 250 mM citrate buffer, the final pH was adjustedto pH 4.5. In the second measurement, the blank sample, 200 μl ofdiluted sample was transferred to a vial that contained 700 μl of waterinstead of pretreated washed wheat straw substrate, and 100 μl of 250 mMcitrate buffer, the final pH was adjusted to pH 4.5. The assay sampleswere incubated for 20 and/or 60 hr at 65° C. After incubation of theassay samples, 100 μl of internal standard solution (20 g/L maleic acid,40 g/L EDTA in D₂O) was added. The amount of glucose released, was basedon the signal at 5.20 ppm, relative to Dimethyl-sila-pentane-sulfonatedetermined by means of 1 D ¹H NMR operating at a proton frequency of 500MHz, using a pulse program with water suppression, at a temperature of27° C. The WSU number was calculated from the data by subtracting by theamount of glucose that was detected in the blank sample from the amountof glucose that was measured in the sample incubated with wheat straw.

The results of the WSU assay are shown in FIG. 3B. In supernatants ofstrains expressing a single cellulase or in empty strains, no WSUactivity could be measured. In A. niger transformants expressing allfour cellulases, e.g. transformant 18 and 19, up to 17 WSU/ml wasmeasured.

The experiment indicates that pGBTOPEBA4, pGBTOPEBA8, pGBFINEBA176 andpGBTOPEBA205 are expressed in A. niger and that A. niger strainsoverexpressing multiple T. emersonii cellulases in maltose-containingmedium are able to degrade pre-treated wheat straw.

Materials and Methods Example 2 Fermentation Medium

Talaromyces Medium 1

Glucose 20 g/L Yeast extract (Difco) 20 g/L Clerol FBA3107 (AF) 4drops/L pH 6.0 Sterilize 20 min at 120° C.Talaromyces Medium 2

Salt fraction 15 g Cellulose 30 g Bacto peptone 7.5 g Grain flour 15 gKH₂PO₄ 10 g CaCl₂•2H20 0.5 g Clerol FBA3107 (AF) 0.4 ml pH 5 WaterAdjust to one liter Sterilize 20 min at 120° C.

Talaromyces medium 3

Salt fraction 15 g Glucose 50 g Bacto peptone 7.5 g KH₂PO₄ 10 gCaCl₂•2H₂0 0.5 g Clerol FBA3107 (AF) 0.4 ml pH 5 Water Adjust to oneliter Sterilize 20 min at 120° C.Spore Batch Preparation

Strains were grown from stocks on Talaromyces agar medium in 10 cmdiameter Petri dishes for 5-7 days at 40° C. Strain stocks were storedat −80° C. in 10% glycerol.

Shake Flask Growth Protocol

Spores were directly inoculated into 500 ml shake flasks containing 100ml of either Talaromyces medium 1 or 2 and incubated at 45° C. at 250rpm in an incubator shaker for 3-4 days.

Sample Preparation

For shake flask cultures, 3 ml of culture broth was transferred to a 12ml disposable tube and centrifuged for 10 min at 5200 g. At least 1 mlof supernatant was harvested.

Protein Analysis

Protein samples were separated under reducing conditions on NuPAGE 4-12%Bis-Tris gel (Invitrogen, Breda, The Netherlands) and stained asindicated. Gels were stained with either InstantBlue (Expedeon,Cambridge, United Kingdom), SimplyBlue safestain (Invitrogen, Breda, TheNetherlands) or Sypro Ruby (Invitrogen, Breda, The Netherlands))according to manufacturer's instructions.

For Western blotting, proteins were transferred to nitrocellulose. Thenitrocellulose filter was blocked with TBST (Tris buffered salinecontaining 0.1% Tween 40) containing 3% skim-milk and incubated for 16hours with anti-FLAG M2 antibody (Sigma, Zwijndrecht, The Netherlands).Blots were washed twice with TBST for 10 minutes and stained withHorse-radish-peroxidase conjugated rabbit-anti-mouse antibody (DAKO,Glostrup, Denmark) for 1 hour. After washing the blots five times withTBST for 10 minutes, proteins were visualized using SuperSignal (Pierce,Rockford, U.S.A).

Wheat Straw Assay (WSU Assay).

Preparation of Pre-Treated, Washed Wheat Straw Substrate.

The washed wheat straw substrate was homogenised using an ultra-turrax,washed, lyophilized and grinded prior to analysis.

Measurement of Cellulase Activity in WSU/ml

Cellulase activity was herein measured in terms of “Wheat Straw Units”(WSU) per milliliter in a Wheat Straw assay (WSU assay). The washedwheat straw substrate was ultraturraxed, washed, lyophilized and grindedprior to analysis.

400 μl of supernatants harvested from shake flask experiments werediluted 16-fold. Duplicate, 200 μl samples were transferred to twosuitable vials: one vial containing 700 μL 3% (w/w) dry matter of thepretreated, washed wheat straw substrate and 100 μl 250 mM citratebuffer, buffered at pH 4.5. The other vial consisted of a blank, wherethe 700 μl 3% (w/w) dry matter pretreated, washed wheat straw substratewas replaced by 700 μl water, with 100 μl 250 mM citrate buffer,buffered at pH 4.5. The assay samples are incubated for 20 and/or 60 hrat 65° C. After incubation of the assay samples, a fixed volume of D₂Ocontaining an internal standard, maleic acid is added. The amount ofsugar released, is based on the signal between 5.25-5.20 ppm, relativeto Dimethyl-sila-pentane-sulfonate determined by means of 1D ¹H NMRoperating at a proton frequency of 500 MHz, using a pulse program withwater suppression, at a temperature of 27° C. The cellulase enzymesolution may contain residual sugars. Therefore, the results of theassay are corrected for the sugar content of the enzyme solution.

Example 2 Overexpression of Multiple Talaromyces emersonii Cellulases inTalaromyces emersonii

Cloning of T emersonii Genes in Expression Vectors

The genes encoding T. emersonii cellobiohydrolase-I (CBHI), T. emersoniibeta-glucanase CEA (EG), and T. emersonii 8-glucosidase (BG) weresynthesised by DNA2.0 (Menlo Park, USA) and cloned as EcoRI/SnaBIfragment into the pGBTOP12 vector, comprising the glucoamylase promoterand terminator sequence, resulting in vector pGBTOPEBA205, pGBTOPEBA8and pGBTOPEBA4, respectively. For cloning purposes, 198 nucleotides ofthe 3′ part of the glucoamylase promoter was also synthesised with thegenes. The amino acid sequences of the T. emersonii cellobiohydrolase-I(CBHI), T. emersonii beta-glucanase CEA (EG), and T. emersoniiβ-glucosidase (BG) are represented by SEQ ID NO: 1, 3, and 5,respectively. The DNA sequences of the genes are represented by SEQ IDNO: 2, 4 and 6, respectively. FIG. 7 represents a map of a pGBTOPEBA205containing the T. emersonii CBHI protein under control of the glaApromoter within vector pGBTOP12. pGBTOPEBA205 is representative forpGBTOPEBA8, which comprises T. emersonii EG, and pGBTOPEBA4, whichcomprises T. emersonii BG.

The gene encoding T. emersonii cellobiohydrolase-II (CBHII), wasobtained from a T. emersonii cDNA library described in patentWO/2001/070998. FIG. 4 represents a map of pGBFINEBA176 containing theT. emersonii CBHII protein under control of the glaA promoter withinvector pGBFIN11. The amino acid sequence and nucleotide sequence arerepresented by SEQ ID NO: 7 and 8, respectively.

Transformation of T. emersonii with Constructs Encoding Cellulases

Transformation of T. emersonii with constructs encoding cellulases wasperformed as described in EXAMPLE 1 of PCT/EP2010/066796. In total, 10μg of DNA was used to co-transform T. emersonii: 1 μg of pAN8-1 and 2 μgof each of the vectors pGBTOPEBA4, pGBTOPEBA8, pGBTOPEBA205 andpGBFINEBA176.

Screening for Transformants Expressing all 4 Cellulases

Transformants were picked from plates and further grown into 96 wellsmicrotiter plates (MTP) containing Talaromyces agar medium for 5 days at40° C. The plates were replica plated using a 96-pin replicator into96-well MTPs containing PDA medium. The MTP plates were incubated for 3days at 40° C. and used to harvest spores for shake flask analysis. Todo this, 100 μl of Talaromyces medium 1 was added to each well and afterresuspending the mixture, 30 μl of spore suspension was used toinoculate 170 μl of Talaromyces medium 1 in MTP plates. The 96-wellplates were incubated in humidity shakers (Infors) for 44° C. at 550rpm, and 80% humidity for 96 hours. Plungerplates were used to push downthe mycelium and, subsequently, approximately 100 μl of supernatant washarvested per well.

Approximately 10 μl of supernatant was analysed for protein expressionusing the E-PAGE 96 Protein electrophoresis system (Invitrogen, Breda,The Netherlands). Gels were stained with SimplyBlue protein staining andtransformants expressing multiple cellulases were selected. Spores ofinteresting transformants were harvested from MTP master plates and usedfor spore batch preparations.

T. emersonii Shake Flask Fermentations and Sample Analysis

T. emersonii transformants expressing one or more cellulases were usedfor shake flask fermentations in Talaromyces medium 2 containing 5% ofglucose. Analysis of protein expression by SDS-PAGE analysis wasperformed. Proteins were visualised using SYPRO Ruby protein straining.

The results of the SYPRO Ruby stained SDS-PAGE gel is presented in FIG.4A. The different transformants expressed different combinations andexpression levels of cellulases. The supernatant of transformant 20(strain 20), contained all 4 cellulases, while, in contrast, nocellulase proteins were observed in the empty strain (FBG142).Therefore, multiple cellulases can simultaneously be overexpressed in T.emersonii in the presence of glucose.

In order to test cellulase activity in T. emersonii transformantsexpressing one or more cellulases, WSU activity was measured insupernatants of an empty strain and the transformants. The results ofthe WSU assay is shown in FIG. 4B. In supernatants harvested after 72hours from cultures of the empty strain grown in medium containingglucose no WSU activity could be measured. In contrast, in transformantsa range of activities could be observed.

Transformant 20 expressing all 4 cellulases showed highest activity:almost 6 WSU/ml, or 5 WSU/ml or more. Transformants 20 and 28 had anactivity of 4 WSU/ml or more, Transformants 20, 28, 6, 64, 33, 11 and 48had an activity of 3 WSU/ml or more, Transformants 20, 28, 6, 64, 33,11, 48, 36, 35 and 1 had an activity of 2.5 WSU/ml or more, andTransformants 20, 28, 6, 64, 33, 11, 48, 36, 35, 1, 43, 53 and 7 had anactivity of 2 WSU/ml or more. All other transformants had an activitywell below 1,5 WSU/ml.

To test whether transformant 20 also produced cellulase activity in theabsence of an inducer, a shake flask fermentation was performed usingTalaromyces medium 3. Supernatants were harvested at day 3, 4 and 5 andanalysed for WSU activity. The results of the WSU assay are shown inTable 2.

TABLE 2 Results of WSU activity measurement in supernatants of an emptystrain and T. emersonii transformant 20 in Talaromyces medium 3.cellulase activity (WSU/ml) Strain Day 3 Day 4 Day 5 Transformant 20(multiple 6.1 7.7 8.1 recombinant cellulases) Empty strain 0.0 0.4 0.9

No cellulase activity was observed in day 3 sups of an empty strain,while some activity was observed at later time-points. In contrast, thetransformant overexpressing multiple cellulases under control of theglaA promoter showed WSU activity at day 3 (6.1 WSU/ml), and theactivity further increased over time.

This experiment shows that T. emersonii transformants comprisingmultiple cellulases under control of the glaA promoter are able toproduce cellulase activity in glucose containing medium with and withoutcellulose. The transformant can be obtained by screening a pool oftransformants that have been transformed with 4 cellulase constructs.

The invention claimed is:
 1. A process for saccharification oflignocellulosic material comprising: (a) optionally, pretreatment oflignocellulosic material, and (b) contacting said lignocellulosicmaterial with an enzyme composition comprising at least four differentTalaromyces enzymes selected from the group consisting of cellulases,hemicellulases, and pectinases, said at least four different Talaromycesenzymes being produced and secreted by a transformed filamentous fungushost cell.
 2. The process of claim 1, wherein the transformedfilamentous fungus host cell is transformed with at least oneheterologous polynucleotide encoding the at least four differentTalaromyces enzymes.
 3. The process of claim 1, wherein the enzymecomposition is an unpurified fermentation broth in which the host cellwas cultured to produce and secrete the at least four differentTalaromyces enzymes, and wherein the unpurified fermentation brothcontains the host cell.
 4. The process of claim 1, wherein the enzymecomposition is a purified fermentation broth in which the host cell wascultured to produce and secrete the at least four different Talaromycesenzymes, and wherein the host cell was removed from the fermentationbroth.
 5. The process of claim 1, wherein the host cell is anAspergillus strain.
 6. The process of claim 1, wherein the host cell isa Talaromyces strain.
 7. The process of claim 1, wherein the enzymecomposition comprises at least cellobiohydrolase-I, β-glucosidase,β-glucanase, and cellobiohydrolase-II.
 8. The process of claim 1,wherein the enzyme composition comprises from 10%-65%cellobiohydrolase-I, from 2%-12% β-glucosidase, from 12%-70%endoglucanase, and from 10%-40% cellobiohydrolase-II.
 9. The process ofclaim 1, wherein the enzyme composition comprises from 10%-60%cellobiohydrolase-I, from 4%-12% β-glucosidase, from 18%-50%endoglucanase, and from 10%-35% cellobiohydrolase-II.